Materials Science and Engineering C 40 (2014) 235–241
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Direct electrochemistry and electrocatalysis of hemoglobin in graphene oxide and ionic liquid composite film Wei Sun a,⁎, Shixing Gong b, Fan Shi a, Lili Cao b, Luyang Ling b, Weizhe Zheng a, Wencheng Wang a a b
College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
i n f o
Article history: Received 15 July 2013 Received in revised form 18 February 2014 Accepted 18 March 2014 Available online 26 March 2014 Keywords: Hemoglobin Graphene oxide Ionic liquid Carbon ionic liquid electrode Electrochemistry
a b s t r a c t In this paper a novel sensing platform based on graphene oxide (GO), ionic liquid (IL) 1-ethyl-3-methylimidazolium tetrafluoroborate and Nafion for the immobilization of hemoglobin (Hb) was adopted with a carbon ionic liquid electrode (CILE) as the substrate electrode, which was denoted as Nafion/Hb–GO–IL/CILE. Spectroscopic results suggested that Hb molecules were not denatured in the composite. A pair of well-defined redox peaks appeared on the cyclic voltammogram, which was attributed to the realization of direct electron transfer of Hb on the electrode. Electrochemical behaviors of Hb entrapped in the film were carefully investigated by cyclic voltammetry with the electrochemical parameters calculated. Based on the catalytic ability of the immobilized Hb, Nafion/Hb–GO–IL/CILE exhibited excellent electrocatalytic behavior towards the reduction of different substrates such as trichloroacetic acid in the concentration range from 0.01 to 40.0 mM with the detection limit as 3.12 μM (3σ), H2O2 in the concentration range from 0.08 to 635.0 μM with the detection limit as 0.0137 μM (3σ) and NaNO2 in the concentration range from 0.5 to 800.0 μM with the detection limit as 0.0104 μM (3σ). So the proposed bioelectrode could be served as a new third-generation electrochemical sensor without mediator. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Studies on the direct electrochemistry of redox proteins with the electrode have attracted great interests in recent years, which have the potential applications in the fields of biosensors, biomedical devices and bioreactors [1]. Because of its well-known structure and commercial availability, hemoglobin (Hb) is often regarded as an ideal model molecule for the investigation. However, most electroactive sites of redox enzymes or proteins are embedded deeply inside the surrounding peptide chain, so it is rather difficult for them to undergo facile redox reactions and exchange electrons with bare electrodes. Therefore, different approaches and materials have been explored for the immobilization of redox enzymes, which can accelerate the direct electron transfer and realize the direct electrochemistry [2]. Due to the unique structure of graphene (GR) with excellent electrical, thermal and mechanical properties, GR and its related composites have been widely investigated recently [3,4]. As a precursor of GR with oxygen-containing functional groups on the basal plane and the sheet edge, graphene oxide (GO) exhibits potential applications in different fields such as electronics, optical materials, solar cells and biosensors [5]. The presence of abundant oxygen-containing functionalities such as epoxide, hydroxyl and carboxylic groups can significantly affect the
⁎ Corresponding author. Tel./fax: +86 898 31381637. E-mail address: [email protected] (W. Sun).
http://dx.doi.org/10.1016/j.msec.2014.03.035 0928-4931/© 2014 Elsevier B.V. All rights reserved.
van der Waals interactions existing between the nanosheets, which render it excellent water dispersible ability. GO can be easily processed in solution to react with different organic and inorganic materials through non-covalent or covalent interactions. Therefore GO is a promising basic nanomaterial for the construction of novel hybrid materials or composites [6]. Also the excellent biocompatibility of GO makes it a good matrix for the immobilization of enzyme, which can not only preserve the native structure and bioactivities of the immobilized enzyme, but also exhibit good electron transfer properties [7]. However, the conductivity of GO is relatively poor due to the unrecovery of the heterogeneous chemical and electronic structure, which limits its electrochemical applications. Ionic liquids (ILs) are compounds consisted entirely of ions that exist in liquid state around room temperature, which have attracted many attentions due to the properties such as extraordinarily high chemical and thermal stability, good ionic conductivity, wide electrochemical windows and good dissolving capability. Among them imidazolium based IL has been proven to be a suitable media for bioelectrocatalysis with different kinds of redox proteins [8]. ILs have also been widely used in the fields of electroanalysis and electrochemical sensors due to their specific electrochemical properties [9,10]. One of the applications is the binder and the modifier in the traditional carbon paste electrode (CPE), and the fabricated electrode is often denoted as carbon ionic liquid electrode (CILE). CILE can act as the working electrode with excellent performances including resistivity towards electrode fouling, high rates of electron transfer and the inherent catalytic activity [11]. Also
236
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
IL can interact with other materials to get the composites, which exhibit the synergistic effects. For examples, IL–carbon nanotube (CNT) or IL– mesoporous carbon had been prepared for the electrode modification, which was further used for the fabrication of electrochemical biosensors [12,13]. Direct electrochemistry of some redox proteins had also been investigated in the IL-based electrochemical systems [14–16]. In this paper a nanocomposite composed of GO and IL was prepared and further utilized as a matrix to immobilize Hb. By using CILE as the substrate electrode and Nafion as the film-forming material, a Nafion/ Hb–GO–IL/CILE was prepared. Due to the specific properties of GO and IL such as good biocompatibility, large surface area, good dispersing properties, fast electron transfer ability and their synergistic effects, the as-prepared enzyme electrode could not only realize the direct electron transfer between Hb and the underling electrode, but also exhibit excellent electrocatalytic performance towards the reduction of different substrates such as trichloroacetic acid (TCA), hydrogen peroxide (H2O2) and NaNO2.
3. Results and discussion
2. Experimental
3.1. FT-IR and UV–vis absorption spectroscopy
2.1. Reagents
FT-IR spectroscopy was used to monitor the possible structural changes of Hb in the GO–IL film. The characteristic amide I (1700–1600 cm−1) and amide II (1620–1500 cm−1) bands of proteins can provide the detailed information on the secondary structure of polypeptide chain. Fig. 1A showed the FT-IR spectra of free Hb (curve a) and Hb–GO–IL composite (curve b). It can be seen that the amide I and II bands of native Hb were located at 1654.8 and 1535.2 cm− 1. After mixing GO, IL and Hb together, the amide I and II bands only shifted slightly to 1656.7 and 1537.2 cm−1. The results demonstrated that the secondary structures of Hb were undisturbed after mixed with GO and IL. The secondary structure of proteins in the composite was further investigated by UV–vis absorption spectroscopy. The band shift may provide conformational information about the denaturation on the tertiary structure of proteins [19]. As shown in Fig. 1B, an intense absorption peak appeared at 407 nm, which was the typical Soret band of free Hb (curve a). While the position of the Soret band was almost unaffected after Hb was immobilized within the GO–IL composite, suggesting that Hb was not denatured. The result can be attributed to the good biocompatibility of GO and IL used.
1-Hexylpyridinium hexafluorophosphate (HPPF 6 , Lanzhou Greenchem. ILS. LICP. CAS., China), 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4, N99%, Lanzhou Greenchem. ILS. LICP. CAS., China), bovine hemoglobin (Hb, MW. 64500, Tianjin Chuanye Biochemical Ltd. Co., China), Nafion (0.5% ethanol solution, Sigma), graphite powder (average particle size 30 μm, Shanghai Colloid Chemical Plant, China) and trichloroacetic acid (TCA, Tianjin Kemiou Chemical Ltd. Co., China) were used as received. GO was synthesized from graphite powder according to Hummers' method [17]. 0.1 M Phosphate buffer solutions (PBS) with various pH values were used as the supporting electrolyte. All the other chemicals were of analytical reagent grade and doubly distilled water was used to prepare all the aqueous solutions. 2.2. Apparatus Electrochemical experiments were performed on a CHI 750B electrochemical workstation (Shanghai CH Instrument, China) with a conventional three-electrode system, which was composed of a modified CILE as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire as the counter electrode. UV–Visible absorption spectrum and Fourier-transform infrared (FTIR) spectrum were obtained on a Cary 50 probe spectrophotometer (Varian Company, Australia) a Tensor 27 FT-IR spectrophotometer (Bruker, Germany), respectively. 2.3. Electrode preparation CILE was fabricated according to the reported method [18]. By carefully mixing 0.8 g of HPPF 6 and 1.6 g of graphite powder in a mortar, a portion of homogeneous paste was packed firmly into a glass tube (Ф = 4.2 mm). The electrical contact was established through a copper wire to the end of the paste in the inner hole of the tube. Then the surface of CILE was polished on a weighing paper just before use. The modifier casted on the electrode surface was prepared with the following procedure. Typically a mixture aqueous solution containing 15.0 mg mL−1 Hb, 0.5 mg mL−1 GO and EMIMBF4 (VIL/Vtotal = 0.5%) was prepared. Then 8 μL of the mixture was evenly dropped onto the surface of CILE and the electrode was left in the air to allow the water evaporated gradually. Finally, 5.0 μL Nafion solution was applied on the electrode surface and dried to get a uniform film. The fabricated electrode was denoted as Nafion/Hb–GO–IL/CILE and kept at 4 °C refrigerator when not use. For comparison other modified electrodes such as
Nafion/GO–IL/CILE, Nafion/Hb/CILE, Nafion/Hb–GO/CILE and Nafion/ Hb–IL/CILE were prepared with the similar procedures.
2.4. Electrochemical measurement Electrochemical measurements were performed at room temperature (20 °C ± 2 °C) by using a CHI 750B electrochemical workstation. The buffer solutions were deoxygenated by bubbling highly purified nitrogen thoroughly for at least 30 min before the experiments and the nitrogen atmosphere environment was kept in the electrochemical cell during the procedure. The three-electrode system was immersed in a 10 mL electrochemical cell containing 0.1 M pH 3.0 PBS and cyclic voltammograms were recorded in the potential range from 0.2 to −0.6 V (vs. SCE) at the scan rate of 100 mV s−1.
3.2. Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) can provide the information of the impedance changes of the electrodes during the modification. 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 0.1 M KCl solution containing 10.0 mM [Fe(CN)6]3−/4− with the frequencies swept from 105 to 10−1 Hz and the results were shown in Fig. 2. The Ret value of CILE (curve b) was obtained as 52.14 Ω, which was due to the presence of high ionic conductive ILs in the carbon paste. On Nafion/ Hb/CILE (curve f) the Ret value increased to 183.7 Ω, indicating that the presence of Hb and Nafion on the electrode surface hindered the electron transfer rate of [Fe(CN)6]3−/4−. While on Nafion/Hb–GO/CILE (curve e) and Nafion/Hb–IL/CILE (curve d), the Ret values decreased to 150.6 Ω and 112.9 Ω, respectively, which could be attributed to the presence of conductive GO and IL in the film. On Nafion/Hb–GO–IL/CILE (curve c), the Ret value further decreased to 74.51 Ω, indicating that the copresence of GO and IL could result in a more conductive composite with the further decrease of the interfacial resistance. While the smallest Ret value of 37.46 Ω (curve a) appeared at Nafion/GO–IL/CILE, indicating that the GO–IL composite exhibited the synergistic effects including the high surface area of GO and high ionic conductive IL, so the resistance of the electrode surface decreased greatly.
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
237
Fig. 1. (A) FT-IR spectra of (a) Hb; (b) Hb–GO–IL composite; (B) UV–vis absorption spectra of Hb in water (a) and Hb–GO–IL mixture (b).
3.3. Direct electrochemistry of the Hb modified electrode Fig. 3 displayed the typical cyclic voltammograms of different modified electrodes in pH 3.0 PBS. No redox peak was observed on Nafion/ GO–IL/CILE, suggesting that this electrode was stable in the studied potential window. On Nafion/Hb/CILE (curve b) a pair of unsymmetric redox peaks appeared. The reduction peak current was bigger than that of the oxidation peak current, indicating a quasi-reversible electron transfer process. After the addition of GO in the film, the redox peak currents increased gradually than that of Nafion/Hb/CILE with a more symmetric peak shape (curve c), which was primarily attributed to the presence of layered GO sheets with high specific surface area and certain conductivity. On Nafion/Hb–IL/CILE (curve d), the redox peak currents were further increased, which was due to the presence of high ionic conductive IL in the film that accelerated the electron transfer rate of Hb. While on Nafion/Hb–GO–IL/CILE (curve e), a pair of welldefined redox peaks appeared with the biggest redox peak currents, which was about 3.89 times higher than that of Nafion/Hb/CILE. The results could be attributed to the synergistic effects of GO and IL in the film. IL exhibits good ionic conductivity and good biocompatibility, while GO can provide large surface area with layered structure. Also GO can interact with IL by the π–π covalent action to give a stable composite and the presence of IL can compensate the poor conductivity of GO. So the direct electron transfer of Hb was realized on the GO–IL composite modified electrode. The reduction peak potential (Epc) and the oxidation peak potential (Epa) were located at −0.221 V and −0.148 V with the peak-to-peak separation (ΔEp) value calculated as 73 mV, and the redox peak currents gave the almost equal values which
Fig. 2. EIS of (a) Nafion/GO–IL/CILE, (b) CILE, (c) Nafion/Hb–GO–IL/CILE, (d) Nafion/ Hb–IL/CILE, (e) Nafion/Hb–GO/CILE and (f) Nafion/Hb/CILE in the presence of 10.0 mM [Fe(CN6)]3-/4- and 0.1 M KCl with the frequencies swept from 105 to 10−1 Hz.
indicated a quasi-reversible electrochemical process. The formal peak potential (E0′), which was calculated from the midpoint of the redox peak potentials, was got as − 0.185 V and this result was the typical characteristic of Hb heme Fe(III)/Fe(II) active center. So the GO–IL composite film on the CILE surface promoted the direct electron transfer efficient of Hb greatly. It is well-known that most of the heme proteins exhibit a pHdependent conformational equilibrium, so the pH of the buffer solution influences the electrochemical reaction of the heme proteins. The effect of buffer pH on the electrochemical response of Nafion/Hb–GO–IL/CILE was investigated in the pH range from 1.0 to 7.0. As shown in Fig. 4A, the increase of buffer pH led to a negative shift of both the redox peak potentials, which indicated that protons were involved in the electrode reaction. The maximum redox peak currents were obtained at pH 3.0 buffer solution, which was selected for the further investigation. As shown in Fig. 4B, the formal peak potential (E0′) was linearly dependent on the buffer pH with the regression equation as E0′(V) = −0.0504 pH– 0.149 (γ = 0.998). The slope value of −50.4 mV pH−1 was close to the theoretical value of −59.0 mV pH−1 [20], indicating that equal amounts of protons and electrons transfer involved in the electrode reaction. 3.4. Effect of scan rate Fig. 5A showed the influence of scan rate on the cyclic voltammetric response of Nafion/Hb–GO–IL/CILE. Along with the increase of scan rate a pair of symmetric redox peaks appeared with the almost equal height of redox peak currents. The linear
Fig. 3. Cyclic voltammograms of (a) Nafion/GO–IL/CILE, (b) Nafion/Hb/CILE, (c) Nafion/ Hb–GO/CILE, (d) Nafion/Hb–IL/CILE and (e) Nafion/Hb–GO–IL/CILE in pH 3.0 PBS at the scan rate of 100 mV s−1.
238
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
Fig. 4. (A) Cyclic voltammograms of Nafion/Hb–GO–IL/CILE in buffer solutions with different pH (a to e: 1.5, 3.0, 4.0, 5.0, 6.0) at the scan rate of 100 mV s−1; (B) the relationship between the formal peak potentials (E0′) and pH.
relationships between the redox peak currents and scan rate were obtained in the range from 50 to 800 mV s− 1 with the two linear regression equations as Ipc(μA) = 87.38 υ (V s− 1) –0.52 (γ = 0.998) and Ipa(μA) = − 83.25υ(V s − 1 ) + 0.80 (γ = 0.998), indicating a typical surface-confined electrochemical behavior. According to Faraday's law: Q = nFAΓ* [21], the surface coverage of the electroactive Hb (Γ*) can be further estimated as 3.04 × 10−9 mol cm−2, which was much larger than that of monolayer coverage (2.0 × 10−11 mol cm−2) [22]. While the total coverage of Hb on the electrode surface was calculated as 1.405 × 10−8 mol cm−2, so 21.60% of the immobilized Hb took part in the electrode reaction. The results demonstrated that the composite was efficiency for the immobilized Hb to exchange electrons. So the presence of GO–IL composite can give a much bigger surface area with layered structures for the several layers of Hb near the electrode surface to undergo the electrochemical reaction. With the increase of scan rate the redox peak potentials also shifted slightly with the increase of ΔEp value gradually. The redox peak potentials exhibited linearly dependant with natural logarithm of scan rate (ln υ) in the range from 50 to 800 mV s− 1 (as shown in Fig. 5B). The regression equations were obtained as Epc(V) = − 0.042lnυ–0.24 (n = 16, γ = 0.998) and Epa(V) = 0.031lnυ–0.13 (n = 16, γ = 0.997), respectively. Then the electrochemical parameters of Hb in the composite could be calculated according to the following Laviron's equations [23,24]: 00
Epc ¼ E −
RT ln ν αnF
ð1Þ
00
Epa ¼ E þ
RT ln ν ð1−αÞnF
logks ¼ αlogð1−αÞ þ ð1−αÞ logα− log
ð2Þ
RT ð1−αÞαnF Δ Ep − nFv 2:3RT
ð3Þ
where α is the electron transfer coefficient, n is the number of electron transferred, υ is the scan rate, E0′ is the formal peak potential, ks is the electron transfer rate constant and ΔEp is the peak-to-peak separation. R, T and F have their conventional meanings. Based on the above equations, the value of n was estimated as 1.07, suggesting that totally one electron was involved in the reaction. The values of α and ks were calculated as 0.57 and 0.92 s− 1. The value of ks is bigger than that of 0.3 s− 1 on GR/Fe3O4/Hb/glassy carbon electrode (GCE) [25] and 0.84 s− 1 on Hb–IL–MWCNT–CPE [26], indicating a fast electron transfer rate. 3.5. Electrocatalytic properties of Nafion/Hb–GO–IL/CILE Electrocatalytic activity of Nafion/Hb–GO–IL/CILE towards various substrates such as TCA, H2O2 and NaNO2 was further investigated by cyclic voltammetry. Fig. 6A illustrated the cyclic voltammograms of Nafion/Hb–GO–IL/CILE with the addition of various concentrations TCA. It can be seen that the reduction peak current at − 0.237 V was greatly increased along with the addition of TCA accompanied by the decrease of the oxidation peak current.
Fig. 5. (A) Cyclic voltammograms of Nafion/Hb–GO–ILHb–GO–IL/CILE in pH 3.0 PBS with different scan rates (a to j: 80, 100, 160, 240, 320, 400, 500, 600, 700, 800 mV s−1); (B) the relationship of anodic and cathodic peak potential against lnυ.
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
239
Fig. 6. (A) Cyclic voltammograms of Nafion/Hb–GO–IL/CILE in the presence of 2.0, 8.0, 12.0, 16.0, 20.0, 24.0, 28.0, 30.0, 34.0, 38.0 mM TCA (from a to j) in pH 3.0 PBS at the scan rate of 100 mV s−1; (B) linear relationship of catalytic reduction peak currents and the TCA concentration.
The result was the typical electrocatalytic reduction process of TCA. Hb based bioelectrode had been reported to exhibit the good catalytic ability to the reduction of organohalides to yield simple hydrocarbons [27]. With the further increase of TCA concentration a new reduction peak appeared at − 0.50 V. The whole electrocatalytic reduction process can be explained with the following equations: Hb hemeFeðIIIÞ þ e→Hb hemeFeðIIÞ
þ
ð4Þ
2 Hb hemeFeðIIÞ þ Cl3 CCOOH þ H →2 Hb hemeFeðIIIÞ þ Cl2 CHCOOH þ Cl
−
ð5Þ Hb hemeFeðIIÞ þ e→Hb hemeFeðIÞ
þ
ð6Þ
−
2 Hb hemeFeðIÞ þ Cl2 CHCOOH þ H →2 Hb hemeFeðIIÞ þ ClCH2 COOH þ Cl
ð7Þ þ
−
2 Hb hemeFeðIÞ þ ClCH2 COOH þ H →2 Hb hemeFeðIIÞ þ CH3 COOH þ Cl
ð8Þ The Hb hemeFe(III) on the electrode surface was reduced to Hb hemeFe(II), which was efficiently converted to the oxidized form of Hb [Hb hemeFe(III)] with the reduction of TCA, and the produced Hb hemeFe(III) was reduced again at the electrode surface in a catalytic cycle. With the further increase of TCA concentration, the second reduction peak at − 0.50 V could be attributed to the formation of a highly reduced form of Hb [Hb hemeFe(I)], which could dechlorinate di and mono chloroacetic acid after the dechlorination of TCA with Hb hemeFe(II) [28]. As shown in Fig. 6B, the catalytic reduction peak currents increased with the TCA concentration in the range from 0.01 to 40.0 mM with the linear regression equation as Iss(μA) = 2.32C (mM) + 16.68 (n = 18, γ = 0.998) and the detection limit was calculated as 3.12 μM (3σ), which was lower than that of the previous results of 0.4 mM on SA–Mb–IL–Fe2 O 3 /CILE [29] and 0.534 mM on CTS/GR–LDH–Hb/ CILE [30]. When the TCA concentration was more than 40.0 mM, the catalytic reduction peak currents began to level off and reached a response plateau, indicating a Michaelis–Menten kinetic mechanism. For a thin film of the immobilized proteins, the maximum current (Imax) measured under the saturated substrate conditions and the apparent Michaelis–Menten constant (KMapp ),
which give an indication of the enzyme–substrate kinetics, can be calculated from the Lineweaver–Burk equation [31]: Kapp 1 1 ¼ þ M Iss Imax Imax c
ð9Þ
where c is the bulk concentration of the substrate. Based on the above equation the K Mapp value was calculated as 0.0244 mM, which was smaller than the reported values of 8.48 mM on CTS/GR–LDH–Hb/CILE [30] and 7.378 mM on Nafion–ZrO 2 –IL– Mb/CILE [32]. The results indicated that the Hb immobilized in the GO–IL composite on the electrode exhibited a high affinity to TCA. Electrocatalytic reduction of H2O2 on Nafion/Hb–GO–IL/CILE was also investigated. With the addition of different amounts of H2O2 into PBS, a significant increase of the reduction peak current was observed at −0.231 V with the decrease of the oxidation peak current, indicating the typical electrocatalytic reduction process to H2O2. Furthermore, the reduction peak current increased linearly with H2O2 concentration in the range from 0.08 to 635.0 μM with the linear regression equation as Iss(μA) = 0.041C (μM) + 18.61 (n = 17, γ = 0.999). The detection limit was calculated as 0.0137 μM (3σ), which was smaller than some previous reported values of 0.4 μM on HRP/GO/Nafion/GCE [33] and 1.17 μM on HRP/GO-MWNT/GCE [34]. The value of K Mapp was further calculated as 13.42 μM, which was lower than the reported values of 0.684 mM on HRP/GO/Nafion/GCE [33] and 0.379 mM on Hb/ZnSnNPs/MWNTs/GCE [35]. The results also indicated an obvious stronger interaction of Hb with H2O 2. The comparison of this Hb modified electrode with other kinds of Hb modified electrodes [36–43] was summarized with the electrochemical data listed in Table 1. It can be seen that this electrode exhibited good electrochemical performance with wider linear range and lower detection limit. Electrocatalytic activity of Nafion/Hb–GO–IL/CILE towards NaNO 2 was further recorded. Experimental results showed that the reduction peak current was linear with the NaNO2 concentration in the range from 0.5 to 800.0 μM and the linear regression equation was got as Iss(μA) = 53.82C (μM) + 13.01 (n = 17, γ = 0.999) with a detection limit of 0.0104 μM (3σ). The result was lower than some previous reported values of 0.2 μM on Hb/ZrO 2/[BMIM]BF4/CHI/GCE [44] and 0.63 μM on Hb/PI/COOH– MWCNTs/GCE [45]. The value of KMapp was further calculated as 10.49 μM, which was lower than the reported values of 7.48 mM on TiO 2 sol–gel/Hb–CSNs/GCE [46] and 4.4 mM on Hb– PVA–GNP–OMIMPF 6 /GCE [47]. Based on the references [48,49],
240
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
Table 1 Comparison of electrochemical parameters of Hb modified electrodes and the analytical performance for H2O2. Hb modified electrodes
Epa (mV)
Epc (mV)
ΔEp (mV)
α
ks (s−1)
Γ⁎ (mol cm−2)
KMapp (μM)
Linear range (μM)
Detection limit (μM)
Ref.
Hb/ZnSnNPs/MWCNTs/GCE Hb–GR–chitosan/GCE Hb/IL/PDDA–G/GCE a GR–Fe3O4–Hb/GCE Hb/GR–ZnO nanosphere/Au Hb/GR–Pt/GCE Nafion–Hb–CoO–IL/CILE Hb/chitosan–[BMIM]PF6–TiO2–GR/GCE Ag@TiO2/GCE Nafion/Hb–GO–IL/CILE
−444 −103 −292 −285 0 −200 −198 −148 / −148
−358 −324 −360 −363 −132 −320 −278 −265 / −221
86 221 68 78 132 120 80 117 / 73
0.48 / / 0.53 0.5 / 0.423 / / 0.57
0.86 / / 0.91 1.0 0.14 1.571 0.73–3.96 / 0.92
3.93 × 10−10 3.1 × 10−10 4.25 × 10−10 / 1.53 × 10−9 4.53 × 10−10 2.47 × 10−9 3.21 × 10−10 / 3.04 × 10−9
379 344 / 3.7 1.46 × 103 540 16 1.245 × 103 / 13.42
0.5–84.00 6.5–230.0 0.2–32.6 1.5–585.0 1.8–2300.0 10.0–1000.0 8.0–140.0 1.0–1170.0 0.83–43.3 0.08–635.0
0.11 0.51 0.4 0.5 0.6 1.0 / 0.3 0.83 0.0137
[35] [36] [37] [38] [39] [40] [41] [42] [43] This work
the possible electrocatalytic reduction scheme can be expressed with the following equations: −
Hb hemeFeðIIIÞ þ e →Hb hemeFeðIIÞ
−
þ
ð10Þ
−
NO2 þ 2H þ 2e →NO þ H2 O
ð11Þ
Hb hemeFeðIIÞ þ NO→Hb hemeFeðIIÞNO
ð12Þ
The electrochemical reduction of Hb hemeFe(III) resulted in Hb hemeFe(II) (Eq. (10)), and NO− 2 can be reduced to NO (Eq. (11)), then the formed NO can be rapidly bound to heme iron of Hb to get Hb hemeFe(II)NO with a irreversible chemical reaction. So the Hb modified electrode can facilitate the reduction of NO− 2 . 3.6. Analytical application The application of this biosensor was evaluated by determining the residual hydrogen peroxide content in a sterilized milk sample, which was purchased from local supermarket. The standard addition method was used to calculate the recovery with the results summarized in Table 2. The samples were also detected by the traditional KMnO4 titration method. No residual H2O2 was found in the milk sample and the results were in agreement with the proposed method. Also the recovery was in the range from 97.3 to 102.9%, showing a satisfactory result.
compounds that maybe present in the biological or environmental samples. With the addition of different kinds of interferents into the buffer solution and the electrochemical responses were further recorded. Then the changes of the peak currents were calculated. Experimental results indicated that the presence of 1.0 mM K+, Zn2+, Ni2+, Mg2+, Cd2+, Cl−, SO2− 4 and 0.1 mM of ascorbic acid, uric acid, cytosine etc. had less influence with the electrochemical responses changed less than ±5%. So the modified electrode exhibited good selectivity for the electrochemical determination. 4. Conclusions A GO–IL nanocomposite was prepared and used for the realization of the direct electrochemistry of Hb on a HPPF6 based CILE. Due to the specific properties of GO, IL and the composite, direct electron transfer of Hb was achieved with fast electron transfer rate. The modified electrode exhibited good electrocatalytic activity toward the reduction of TCA, H2O2 and NaNO2 with wider linear range and lower detection limit. Therefore this approach provided a new strategy for the development of the third-generation electrochemical biosensors with the advantages including good stability, high electrocatalytic ability and simply preparation procedure. Acknowledgment We acknowledge the financial support provide by the National Natural Science Foundation of China (21075071, 21365010), the Natural Science Foundation of Hainan Province (213015) and the Foundation of Hainan Normal University.
3.7. Reproducibility, stability and selectivity References The reproducibility, stability and selectivity of Nafion/Hb–GO–IL/CILE were investigated. Five independent determinations to a 20.0 mM TCA solution showed a relative standard deviation (RSD) of 2.19%, indicating the modified electrode possessed an acceptable reproducibility. To examine the stability, the response of the Nafion/Hb–GO–IL/CILE was examined with respect to the storage time when not in use. The current value decreased for 3.8% and 5.4% of the initial response after 14 days and 30 days storage, respectively, demonstrating the excellent stability of this biosensor. Thus, Nafion/Hb–GO–IL/CILE exhibited good stability for the electrochemical detection in general. The selectivity of Nafion/ Hb–GO–IL/CILE was evaluated by examining the influence of some
Table 2 Detection results of H2O2 in sterilized milk sample (n = 3). Samples
Detected
Added (μM)
Found (μM)
RSD (%)
Recovery (%)
1 2 3 4
0 0 0 0
10.0 30.0 60.0 90.0
9.73 29.6 61.8 88.3
2.16 1.86 3.13 2.39
97.3 98.5 102.9 98.2
[1] F.A. Armstrong, H.A.O. Hill, N.J. Walton, Acc. Chem. Res. 21 (1988) 407–413. [2] J.F. Rusling, Acc. Chem. Res. 31 (1998) 363–369. [3] N.O. Weiss, H.L. Zhou, L. Liao, Y. Liu, S. Jiang, Y. Huang, X.F. Duan, Adv. Mater. 24 (2012) 5782–5825. [4] V. Singh, D. Joung, L. Zhai, S. Das, S.I. Khondaker, S. Seal, Prog. Mater. Sci. 56 (2011) 1178–1271. [5] D. Chen, H.B. Feng, J.H. Li, Chem. Rev. 112 (2012) 6027–6053. [6] D.R. Dreyer, S.J. Park, C.W. Bielawski, R.S. Ruoff, Chem. Soc. Rev. 39 (2010) 228–240. [7] Y. Liu, D.S. Yu, C. Zeng, Z.C. Miao, L.M. Dai, Langmuir 26 (2010) 6158–6160. [8] J.A. Laszlo, D.L. Compton, J. Mol. Catal. B Enzym. 18 (2002) 109–120. [9] W. Sun, R.F. Gao, K. Jiao, Chin. J. Anal. Chem. 35 (2007) 1813–1819. [10] J.A.M. Shiddiky, A.A.J. Torriero, Biosens. Bioelectron. 26 (2011) 1775–1787. [11] N. Maleki, A. Safavi, F. Tajabadi, Anal. Chem. 78 (2006) 3820–3826. [12] R.T. Kachoosangi, M.M. Musameh, I.A. Yousef, J.M. Yousef, S.M. Kanan, L. Xiao, S.G. Davies, A. Russell, R.G. Compton, Anal. Chem. 81 (2009) 435–442. [13] W. Sun, C.X. Guo, Z.H. Zhu, C.M. Li, Electrochem. Commun. 11 (2009) 2105–2108. [14] K.J. Huang, J.Y. Sun, D.J. Niu, W.Z. Xie, W. Wang, Colloid Surf. B 78 (2010) 69–74. [15] C.X. Ruan, T.T. Li, Q.J. Niu, M. Lu, J. Lou, W.M. Gao, W. Sun, Electrochim. Acta 64 (2012) 183–189. [16] R. Li, C.X. Liu, M. Ma, Z.G. Wang, G.Q. Zhan, B.H. Li, X. Wang, H.F. Fang, H.J. Zhang, C.Y. Li, Electrochim. Acta 95 (2013) 71–79. [17] W.S. Hummers, R.E. Offeman, J. Am. Chem. Soc. 80 (1958) 1339–1340. [18] T.R. Zhan, Y.Q. Guo, L. Xu, W.L. Zhang, W. Sun, W.G. Hou, Talanta 94 (2012) 189–194. [19] P. George, C. Hanania, Biochem. J. 55 (1953) 236–243. [20] R.S. Nicholson, Anal. Chem. 37 (1965) 1351–1352.
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241 [21] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 1980. [22] S.F. Wang, T. Chen, Z.L. Zhang, X.C. Shen, Z.X. Lu, D.W. Pang, K.Y. Wong, Langmuir 21 (2005) 9260–9266. [23] E. Laviron, J. Electroanal. Chem. 52 (1974) 355–359. [24] E. Laviron, J. Electroanal. Chem. 101 (1979) 19–28. [25] Y.Q. Wang, H.J. Zhang, D. Yao, J.J. Pu, Y. Zhang, X.R. Gao, Y.M. Sun, J. Solid State Electrochem. 17 (2013) 881–887. [26] W. Wei, H.H. Jin, G.C. Zhao, Microchim. Acta 164 (2009) 167–171. [27] A.A. Ordaz, F. Bedioui, Sens. Actuators B 59 (1999) 128–133. [28] X. Ma, X.J. Liu, H. Xiao, G.X. Li, Biosens. Bioelectron. 20 (2005) 1836–1842. [29] T.R. Zhan, M.Y. Xi, Y. Wang, W. Sun, W.G. Hou, J. Colloid Interface Sci. 346 (2010) 188–193. [30] W. Sun, Y.Q. Guo, Y.P. Lu, A.H. Hu, F. Shi, T.T. Li, Z.F. Sun, Electrochim. Acta 91 (2013) 130–136. [31] R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198–1205. [32] C.X. Ruan, T.T. Li, X.M. Ju, H.J. Liu, J. Lou, W.M. Gao, W. Sun, J. Solid State Electrochem. 16 (2012) 3661–3666. [33] L.L. Zhang, H.H. Cheng, H.M. Zhang, L.T. Qu, Electrochim. Acta 65 (2012) 122–126. [34] Q. Zhang, S.J. Yang, J. Zhang, L. Zhang, P.L. Kang, J.H. Li, J.W. Xu, H. Zhou, X.M. Song, Nanotechnology 22 (2011) 494010–494016. [35] A.L. Sun, H.Y. Zhao, J.B. Zheng, Talanta 88 (2012) 259–264. [36] H.F. Xu, H. Dai, G.N. Chen, Talanta 81 (2010) 334–338. [37] K.P. Liu, J.J. Zhang, G.H. Yang, C.M. Wang, J.J. Zhu, Electrochem. Commun. 12 (2010) 402–405. [38] Y.P. He, Q.L. Sheng, J.B. Zhen, M.Z. Wang, B. Liu, Electrochim. Acta 56 (2011) 2471–2476. [39] J. Xu, C.H. Liu, Z.F. Wu, Microchim. Acta 172 (2011) 425–430. [40] X.M. Feng, R. Li, C.H. Hu, W.H. Hou, J. Electroanal. Chem. 657 (2011) 28–33. [41] Z.H. Zhu, X. Li, Y. Zeng, W. Sun, W.M. Zhu, X.T. Huang, J. Phys. Chem. C 115 (2011) 12547–12553. [42] J.Y. Sun, K.J. Huang, S.F. Zhao, Y. Fan, Z.W. Wu, Bioelectrochemistry 82 (2011) 125–130.
[43] [44] [45] [46] [47] [48] [49]
241
M.M. Khan, S.A. Ansari, J. Lee, M.H. Cho, Mater. Sci. Eng. C 33 (2013) 4692–4699. Y.H. Ma, G.Q. Zhan, M. Ma, X. Wang, C.Y. Li, Bioelectrochemistry 84 (2012) 6–10. H.H. Kou, L.P. Jia, C.M. Wang, W.C. Ye, Electroanalysis 24 (2012) 1799–1803. S. Zhao, K. Zhang, Y.Y. Sun, C.Q. Sun, Bioelectrochemistry 69 (2006) 10–15. Y.F. Zhang, R. Yan, F.Q. Zhao, B.Z. Zeng, Colloid Surf. B 71 (2009) 288–292. J.N. Younathan, K.S. Wood, T.J. Meyer, Inorg. Chem. 31 (1992) 3280–3285. W.W. Yang, Y. Bai, Y.C. Li, C.Q. Sun, Anal. Bioanal. Chem. 382 (2005) 44–50.
Wei Sun is a professor in the College of Chemistry and Chemical Engineering, Hainan Normal University. He received his PhD in analytical chemistry from Ocean University of China in 2002. His current interests are bioelectroanalysis. Shixing Gong is a master candidate in the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology. Fan Shi is a master candidate in the College of Chemistry and Chemical Engineering, Hainan Normal University. Lili Cao is a master candidate in the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology. Luyang Ling is an undergraduate student in the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology. Weizhe Zheng is an undergraduate student in the College of Chemistry and Chemical Engineering, Hainan Normal University. Wencheng Wang is a master candidate in the College of Chemistry and Chemical Engineering, Hainan Normal University.
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Direct electrochemistry and electrocatalysis of hemoglobin in graphene oxide and ionic liquid composite film Wei Sun a,⁎, Shixing Gong b, Fan Shi a, Lili Cao b, Luyang Ling b, Weizhe Zheng a, Wencheng Wang a a b
College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
i n f o
Article history: Received 15 July 2013 Received in revised form 18 February 2014 Accepted 18 March 2014 Available online 26 March 2014 Keywords: Hemoglobin Graphene oxide Ionic liquid Carbon ionic liquid electrode Electrochemistry
a b s t r a c t In this paper a novel sensing platform based on graphene oxide (GO), ionic liquid (IL) 1-ethyl-3-methylimidazolium tetrafluoroborate and Nafion for the immobilization of hemoglobin (Hb) was adopted with a carbon ionic liquid electrode (CILE) as the substrate electrode, which was denoted as Nafion/Hb–GO–IL/CILE. Spectroscopic results suggested that Hb molecules were not denatured in the composite. A pair of well-defined redox peaks appeared on the cyclic voltammogram, which was attributed to the realization of direct electron transfer of Hb on the electrode. Electrochemical behaviors of Hb entrapped in the film were carefully investigated by cyclic voltammetry with the electrochemical parameters calculated. Based on the catalytic ability of the immobilized Hb, Nafion/Hb–GO–IL/CILE exhibited excellent electrocatalytic behavior towards the reduction of different substrates such as trichloroacetic acid in the concentration range from 0.01 to 40.0 mM with the detection limit as 3.12 μM (3σ), H2O2 in the concentration range from 0.08 to 635.0 μM with the detection limit as 0.0137 μM (3σ) and NaNO2 in the concentration range from 0.5 to 800.0 μM with the detection limit as 0.0104 μM (3σ). So the proposed bioelectrode could be served as a new third-generation electrochemical sensor without mediator. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Studies on the direct electrochemistry of redox proteins with the electrode have attracted great interests in recent years, which have the potential applications in the fields of biosensors, biomedical devices and bioreactors [1]. Because of its well-known structure and commercial availability, hemoglobin (Hb) is often regarded as an ideal model molecule for the investigation. However, most electroactive sites of redox enzymes or proteins are embedded deeply inside the surrounding peptide chain, so it is rather difficult for them to undergo facile redox reactions and exchange electrons with bare electrodes. Therefore, different approaches and materials have been explored for the immobilization of redox enzymes, which can accelerate the direct electron transfer and realize the direct electrochemistry [2]. Due to the unique structure of graphene (GR) with excellent electrical, thermal and mechanical properties, GR and its related composites have been widely investigated recently [3,4]. As a precursor of GR with oxygen-containing functional groups on the basal plane and the sheet edge, graphene oxide (GO) exhibits potential applications in different fields such as electronics, optical materials, solar cells and biosensors [5]. The presence of abundant oxygen-containing functionalities such as epoxide, hydroxyl and carboxylic groups can significantly affect the
⁎ Corresponding author. Tel./fax: +86 898 31381637. E-mail address: [email protected] (W. Sun).
http://dx.doi.org/10.1016/j.msec.2014.03.035 0928-4931/© 2014 Elsevier B.V. All rights reserved.
van der Waals interactions existing between the nanosheets, which render it excellent water dispersible ability. GO can be easily processed in solution to react with different organic and inorganic materials through non-covalent or covalent interactions. Therefore GO is a promising basic nanomaterial for the construction of novel hybrid materials or composites [6]. Also the excellent biocompatibility of GO makes it a good matrix for the immobilization of enzyme, which can not only preserve the native structure and bioactivities of the immobilized enzyme, but also exhibit good electron transfer properties [7]. However, the conductivity of GO is relatively poor due to the unrecovery of the heterogeneous chemical and electronic structure, which limits its electrochemical applications. Ionic liquids (ILs) are compounds consisted entirely of ions that exist in liquid state around room temperature, which have attracted many attentions due to the properties such as extraordinarily high chemical and thermal stability, good ionic conductivity, wide electrochemical windows and good dissolving capability. Among them imidazolium based IL has been proven to be a suitable media for bioelectrocatalysis with different kinds of redox proteins [8]. ILs have also been widely used in the fields of electroanalysis and electrochemical sensors due to their specific electrochemical properties [9,10]. One of the applications is the binder and the modifier in the traditional carbon paste electrode (CPE), and the fabricated electrode is often denoted as carbon ionic liquid electrode (CILE). CILE can act as the working electrode with excellent performances including resistivity towards electrode fouling, high rates of electron transfer and the inherent catalytic activity [11]. Also
236
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
IL can interact with other materials to get the composites, which exhibit the synergistic effects. For examples, IL–carbon nanotube (CNT) or IL– mesoporous carbon had been prepared for the electrode modification, which was further used for the fabrication of electrochemical biosensors [12,13]. Direct electrochemistry of some redox proteins had also been investigated in the IL-based electrochemical systems [14–16]. In this paper a nanocomposite composed of GO and IL was prepared and further utilized as a matrix to immobilize Hb. By using CILE as the substrate electrode and Nafion as the film-forming material, a Nafion/ Hb–GO–IL/CILE was prepared. Due to the specific properties of GO and IL such as good biocompatibility, large surface area, good dispersing properties, fast electron transfer ability and their synergistic effects, the as-prepared enzyme electrode could not only realize the direct electron transfer between Hb and the underling electrode, but also exhibit excellent electrocatalytic performance towards the reduction of different substrates such as trichloroacetic acid (TCA), hydrogen peroxide (H2O2) and NaNO2.
3. Results and discussion
2. Experimental
3.1. FT-IR and UV–vis absorption spectroscopy
2.1. Reagents
FT-IR spectroscopy was used to monitor the possible structural changes of Hb in the GO–IL film. The characteristic amide I (1700–1600 cm−1) and amide II (1620–1500 cm−1) bands of proteins can provide the detailed information on the secondary structure of polypeptide chain. Fig. 1A showed the FT-IR spectra of free Hb (curve a) and Hb–GO–IL composite (curve b). It can be seen that the amide I and II bands of native Hb were located at 1654.8 and 1535.2 cm− 1. After mixing GO, IL and Hb together, the amide I and II bands only shifted slightly to 1656.7 and 1537.2 cm−1. The results demonstrated that the secondary structures of Hb were undisturbed after mixed with GO and IL. The secondary structure of proteins in the composite was further investigated by UV–vis absorption spectroscopy. The band shift may provide conformational information about the denaturation on the tertiary structure of proteins [19]. As shown in Fig. 1B, an intense absorption peak appeared at 407 nm, which was the typical Soret band of free Hb (curve a). While the position of the Soret band was almost unaffected after Hb was immobilized within the GO–IL composite, suggesting that Hb was not denatured. The result can be attributed to the good biocompatibility of GO and IL used.
1-Hexylpyridinium hexafluorophosphate (HPPF 6 , Lanzhou Greenchem. ILS. LICP. CAS., China), 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4, N99%, Lanzhou Greenchem. ILS. LICP. CAS., China), bovine hemoglobin (Hb, MW. 64500, Tianjin Chuanye Biochemical Ltd. Co., China), Nafion (0.5% ethanol solution, Sigma), graphite powder (average particle size 30 μm, Shanghai Colloid Chemical Plant, China) and trichloroacetic acid (TCA, Tianjin Kemiou Chemical Ltd. Co., China) were used as received. GO was synthesized from graphite powder according to Hummers' method [17]. 0.1 M Phosphate buffer solutions (PBS) with various pH values were used as the supporting electrolyte. All the other chemicals were of analytical reagent grade and doubly distilled water was used to prepare all the aqueous solutions. 2.2. Apparatus Electrochemical experiments were performed on a CHI 750B electrochemical workstation (Shanghai CH Instrument, China) with a conventional three-electrode system, which was composed of a modified CILE as the working electrode, a saturated calomel electrode (SCE) as the reference electrode and a platinum wire as the counter electrode. UV–Visible absorption spectrum and Fourier-transform infrared (FTIR) spectrum were obtained on a Cary 50 probe spectrophotometer (Varian Company, Australia) a Tensor 27 FT-IR spectrophotometer (Bruker, Germany), respectively. 2.3. Electrode preparation CILE was fabricated according to the reported method [18]. By carefully mixing 0.8 g of HPPF 6 and 1.6 g of graphite powder in a mortar, a portion of homogeneous paste was packed firmly into a glass tube (Ф = 4.2 mm). The electrical contact was established through a copper wire to the end of the paste in the inner hole of the tube. Then the surface of CILE was polished on a weighing paper just before use. The modifier casted on the electrode surface was prepared with the following procedure. Typically a mixture aqueous solution containing 15.0 mg mL−1 Hb, 0.5 mg mL−1 GO and EMIMBF4 (VIL/Vtotal = 0.5%) was prepared. Then 8 μL of the mixture was evenly dropped onto the surface of CILE and the electrode was left in the air to allow the water evaporated gradually. Finally, 5.0 μL Nafion solution was applied on the electrode surface and dried to get a uniform film. The fabricated electrode was denoted as Nafion/Hb–GO–IL/CILE and kept at 4 °C refrigerator when not use. For comparison other modified electrodes such as
Nafion/GO–IL/CILE, Nafion/Hb/CILE, Nafion/Hb–GO/CILE and Nafion/ Hb–IL/CILE were prepared with the similar procedures.
2.4. Electrochemical measurement Electrochemical measurements were performed at room temperature (20 °C ± 2 °C) by using a CHI 750B electrochemical workstation. The buffer solutions were deoxygenated by bubbling highly purified nitrogen thoroughly for at least 30 min before the experiments and the nitrogen atmosphere environment was kept in the electrochemical cell during the procedure. The three-electrode system was immersed in a 10 mL electrochemical cell containing 0.1 M pH 3.0 PBS and cyclic voltammograms were recorded in the potential range from 0.2 to −0.6 V (vs. SCE) at the scan rate of 100 mV s−1.
3.2. Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) can provide the information of the impedance changes of the electrodes during the modification. 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 0.1 M KCl solution containing 10.0 mM [Fe(CN)6]3−/4− with the frequencies swept from 105 to 10−1 Hz and the results were shown in Fig. 2. The Ret value of CILE (curve b) was obtained as 52.14 Ω, which was due to the presence of high ionic conductive ILs in the carbon paste. On Nafion/ Hb/CILE (curve f) the Ret value increased to 183.7 Ω, indicating that the presence of Hb and Nafion on the electrode surface hindered the electron transfer rate of [Fe(CN)6]3−/4−. While on Nafion/Hb–GO/CILE (curve e) and Nafion/Hb–IL/CILE (curve d), the Ret values decreased to 150.6 Ω and 112.9 Ω, respectively, which could be attributed to the presence of conductive GO and IL in the film. On Nafion/Hb–GO–IL/CILE (curve c), the Ret value further decreased to 74.51 Ω, indicating that the copresence of GO and IL could result in a more conductive composite with the further decrease of the interfacial resistance. While the smallest Ret value of 37.46 Ω (curve a) appeared at Nafion/GO–IL/CILE, indicating that the GO–IL composite exhibited the synergistic effects including the high surface area of GO and high ionic conductive IL, so the resistance of the electrode surface decreased greatly.
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
237
Fig. 1. (A) FT-IR spectra of (a) Hb; (b) Hb–GO–IL composite; (B) UV–vis absorption spectra of Hb in water (a) and Hb–GO–IL mixture (b).
3.3. Direct electrochemistry of the Hb modified electrode Fig. 3 displayed the typical cyclic voltammograms of different modified electrodes in pH 3.0 PBS. No redox peak was observed on Nafion/ GO–IL/CILE, suggesting that this electrode was stable in the studied potential window. On Nafion/Hb/CILE (curve b) a pair of unsymmetric redox peaks appeared. The reduction peak current was bigger than that of the oxidation peak current, indicating a quasi-reversible electron transfer process. After the addition of GO in the film, the redox peak currents increased gradually than that of Nafion/Hb/CILE with a more symmetric peak shape (curve c), which was primarily attributed to the presence of layered GO sheets with high specific surface area and certain conductivity. On Nafion/Hb–IL/CILE (curve d), the redox peak currents were further increased, which was due to the presence of high ionic conductive IL in the film that accelerated the electron transfer rate of Hb. While on Nafion/Hb–GO–IL/CILE (curve e), a pair of welldefined redox peaks appeared with the biggest redox peak currents, which was about 3.89 times higher than that of Nafion/Hb/CILE. The results could be attributed to the synergistic effects of GO and IL in the film. IL exhibits good ionic conductivity and good biocompatibility, while GO can provide large surface area with layered structure. Also GO can interact with IL by the π–π covalent action to give a stable composite and the presence of IL can compensate the poor conductivity of GO. So the direct electron transfer of Hb was realized on the GO–IL composite modified electrode. The reduction peak potential (Epc) and the oxidation peak potential (Epa) were located at −0.221 V and −0.148 V with the peak-to-peak separation (ΔEp) value calculated as 73 mV, and the redox peak currents gave the almost equal values which
Fig. 2. EIS of (a) Nafion/GO–IL/CILE, (b) CILE, (c) Nafion/Hb–GO–IL/CILE, (d) Nafion/ Hb–IL/CILE, (e) Nafion/Hb–GO/CILE and (f) Nafion/Hb/CILE in the presence of 10.0 mM [Fe(CN6)]3-/4- and 0.1 M KCl with the frequencies swept from 105 to 10−1 Hz.
indicated a quasi-reversible electrochemical process. The formal peak potential (E0′), which was calculated from the midpoint of the redox peak potentials, was got as − 0.185 V and this result was the typical characteristic of Hb heme Fe(III)/Fe(II) active center. So the GO–IL composite film on the CILE surface promoted the direct electron transfer efficient of Hb greatly. It is well-known that most of the heme proteins exhibit a pHdependent conformational equilibrium, so the pH of the buffer solution influences the electrochemical reaction of the heme proteins. The effect of buffer pH on the electrochemical response of Nafion/Hb–GO–IL/CILE was investigated in the pH range from 1.0 to 7.0. As shown in Fig. 4A, the increase of buffer pH led to a negative shift of both the redox peak potentials, which indicated that protons were involved in the electrode reaction. The maximum redox peak currents were obtained at pH 3.0 buffer solution, which was selected for the further investigation. As shown in Fig. 4B, the formal peak potential (E0′) was linearly dependent on the buffer pH with the regression equation as E0′(V) = −0.0504 pH– 0.149 (γ = 0.998). The slope value of −50.4 mV pH−1 was close to the theoretical value of −59.0 mV pH−1 [20], indicating that equal amounts of protons and electrons transfer involved in the electrode reaction. 3.4. Effect of scan rate Fig. 5A showed the influence of scan rate on the cyclic voltammetric response of Nafion/Hb–GO–IL/CILE. Along with the increase of scan rate a pair of symmetric redox peaks appeared with the almost equal height of redox peak currents. The linear
Fig. 3. Cyclic voltammograms of (a) Nafion/GO–IL/CILE, (b) Nafion/Hb/CILE, (c) Nafion/ Hb–GO/CILE, (d) Nafion/Hb–IL/CILE and (e) Nafion/Hb–GO–IL/CILE in pH 3.0 PBS at the scan rate of 100 mV s−1.
238
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
Fig. 4. (A) Cyclic voltammograms of Nafion/Hb–GO–IL/CILE in buffer solutions with different pH (a to e: 1.5, 3.0, 4.0, 5.0, 6.0) at the scan rate of 100 mV s−1; (B) the relationship between the formal peak potentials (E0′) and pH.
relationships between the redox peak currents and scan rate were obtained in the range from 50 to 800 mV s− 1 with the two linear regression equations as Ipc(μA) = 87.38 υ (V s− 1) –0.52 (γ = 0.998) and Ipa(μA) = − 83.25υ(V s − 1 ) + 0.80 (γ = 0.998), indicating a typical surface-confined electrochemical behavior. According to Faraday's law: Q = nFAΓ* [21], the surface coverage of the electroactive Hb (Γ*) can be further estimated as 3.04 × 10−9 mol cm−2, which was much larger than that of monolayer coverage (2.0 × 10−11 mol cm−2) [22]. While the total coverage of Hb on the electrode surface was calculated as 1.405 × 10−8 mol cm−2, so 21.60% of the immobilized Hb took part in the electrode reaction. The results demonstrated that the composite was efficiency for the immobilized Hb to exchange electrons. So the presence of GO–IL composite can give a much bigger surface area with layered structures for the several layers of Hb near the electrode surface to undergo the electrochemical reaction. With the increase of scan rate the redox peak potentials also shifted slightly with the increase of ΔEp value gradually. The redox peak potentials exhibited linearly dependant with natural logarithm of scan rate (ln υ) in the range from 50 to 800 mV s− 1 (as shown in Fig. 5B). The regression equations were obtained as Epc(V) = − 0.042lnυ–0.24 (n = 16, γ = 0.998) and Epa(V) = 0.031lnυ–0.13 (n = 16, γ = 0.997), respectively. Then the electrochemical parameters of Hb in the composite could be calculated according to the following Laviron's equations [23,24]: 00
Epc ¼ E −
RT ln ν αnF
ð1Þ
00
Epa ¼ E þ
RT ln ν ð1−αÞnF
logks ¼ αlogð1−αÞ þ ð1−αÞ logα− log
ð2Þ
RT ð1−αÞαnF Δ Ep − nFv 2:3RT
ð3Þ
where α is the electron transfer coefficient, n is the number of electron transferred, υ is the scan rate, E0′ is the formal peak potential, ks is the electron transfer rate constant and ΔEp is the peak-to-peak separation. R, T and F have their conventional meanings. Based on the above equations, the value of n was estimated as 1.07, suggesting that totally one electron was involved in the reaction. The values of α and ks were calculated as 0.57 and 0.92 s− 1. The value of ks is bigger than that of 0.3 s− 1 on GR/Fe3O4/Hb/glassy carbon electrode (GCE) [25] and 0.84 s− 1 on Hb–IL–MWCNT–CPE [26], indicating a fast electron transfer rate. 3.5. Electrocatalytic properties of Nafion/Hb–GO–IL/CILE Electrocatalytic activity of Nafion/Hb–GO–IL/CILE towards various substrates such as TCA, H2O2 and NaNO2 was further investigated by cyclic voltammetry. Fig. 6A illustrated the cyclic voltammograms of Nafion/Hb–GO–IL/CILE with the addition of various concentrations TCA. It can be seen that the reduction peak current at − 0.237 V was greatly increased along with the addition of TCA accompanied by the decrease of the oxidation peak current.
Fig. 5. (A) Cyclic voltammograms of Nafion/Hb–GO–ILHb–GO–IL/CILE in pH 3.0 PBS with different scan rates (a to j: 80, 100, 160, 240, 320, 400, 500, 600, 700, 800 mV s−1); (B) the relationship of anodic and cathodic peak potential against lnυ.
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
239
Fig. 6. (A) Cyclic voltammograms of Nafion/Hb–GO–IL/CILE in the presence of 2.0, 8.0, 12.0, 16.0, 20.0, 24.0, 28.0, 30.0, 34.0, 38.0 mM TCA (from a to j) in pH 3.0 PBS at the scan rate of 100 mV s−1; (B) linear relationship of catalytic reduction peak currents and the TCA concentration.
The result was the typical electrocatalytic reduction process of TCA. Hb based bioelectrode had been reported to exhibit the good catalytic ability to the reduction of organohalides to yield simple hydrocarbons [27]. With the further increase of TCA concentration a new reduction peak appeared at − 0.50 V. The whole electrocatalytic reduction process can be explained with the following equations: Hb hemeFeðIIIÞ þ e→Hb hemeFeðIIÞ
þ
ð4Þ
2 Hb hemeFeðIIÞ þ Cl3 CCOOH þ H →2 Hb hemeFeðIIIÞ þ Cl2 CHCOOH þ Cl
−
ð5Þ Hb hemeFeðIIÞ þ e→Hb hemeFeðIÞ
þ
ð6Þ
−
2 Hb hemeFeðIÞ þ Cl2 CHCOOH þ H →2 Hb hemeFeðIIÞ þ ClCH2 COOH þ Cl
ð7Þ þ
−
2 Hb hemeFeðIÞ þ ClCH2 COOH þ H →2 Hb hemeFeðIIÞ þ CH3 COOH þ Cl
ð8Þ The Hb hemeFe(III) on the electrode surface was reduced to Hb hemeFe(II), which was efficiently converted to the oxidized form of Hb [Hb hemeFe(III)] with the reduction of TCA, and the produced Hb hemeFe(III) was reduced again at the electrode surface in a catalytic cycle. With the further increase of TCA concentration, the second reduction peak at − 0.50 V could be attributed to the formation of a highly reduced form of Hb [Hb hemeFe(I)], which could dechlorinate di and mono chloroacetic acid after the dechlorination of TCA with Hb hemeFe(II) [28]. As shown in Fig. 6B, the catalytic reduction peak currents increased with the TCA concentration in the range from 0.01 to 40.0 mM with the linear regression equation as Iss(μA) = 2.32C (mM) + 16.68 (n = 18, γ = 0.998) and the detection limit was calculated as 3.12 μM (3σ), which was lower than that of the previous results of 0.4 mM on SA–Mb–IL–Fe2 O 3 /CILE [29] and 0.534 mM on CTS/GR–LDH–Hb/ CILE [30]. When the TCA concentration was more than 40.0 mM, the catalytic reduction peak currents began to level off and reached a response plateau, indicating a Michaelis–Menten kinetic mechanism. For a thin film of the immobilized proteins, the maximum current (Imax) measured under the saturated substrate conditions and the apparent Michaelis–Menten constant (KMapp ),
which give an indication of the enzyme–substrate kinetics, can be calculated from the Lineweaver–Burk equation [31]: Kapp 1 1 ¼ þ M Iss Imax Imax c
ð9Þ
where c is the bulk concentration of the substrate. Based on the above equation the K Mapp value was calculated as 0.0244 mM, which was smaller than the reported values of 8.48 mM on CTS/GR–LDH–Hb/CILE [30] and 7.378 mM on Nafion–ZrO 2 –IL– Mb/CILE [32]. The results indicated that the Hb immobilized in the GO–IL composite on the electrode exhibited a high affinity to TCA. Electrocatalytic reduction of H2O2 on Nafion/Hb–GO–IL/CILE was also investigated. With the addition of different amounts of H2O2 into PBS, a significant increase of the reduction peak current was observed at −0.231 V with the decrease of the oxidation peak current, indicating the typical electrocatalytic reduction process to H2O2. Furthermore, the reduction peak current increased linearly with H2O2 concentration in the range from 0.08 to 635.0 μM with the linear regression equation as Iss(μA) = 0.041C (μM) + 18.61 (n = 17, γ = 0.999). The detection limit was calculated as 0.0137 μM (3σ), which was smaller than some previous reported values of 0.4 μM on HRP/GO/Nafion/GCE [33] and 1.17 μM on HRP/GO-MWNT/GCE [34]. The value of K Mapp was further calculated as 13.42 μM, which was lower than the reported values of 0.684 mM on HRP/GO/Nafion/GCE [33] and 0.379 mM on Hb/ZnSnNPs/MWNTs/GCE [35]. The results also indicated an obvious stronger interaction of Hb with H2O 2. The comparison of this Hb modified electrode with other kinds of Hb modified electrodes [36–43] was summarized with the electrochemical data listed in Table 1. It can be seen that this electrode exhibited good electrochemical performance with wider linear range and lower detection limit. Electrocatalytic activity of Nafion/Hb–GO–IL/CILE towards NaNO 2 was further recorded. Experimental results showed that the reduction peak current was linear with the NaNO2 concentration in the range from 0.5 to 800.0 μM and the linear regression equation was got as Iss(μA) = 53.82C (μM) + 13.01 (n = 17, γ = 0.999) with a detection limit of 0.0104 μM (3σ). The result was lower than some previous reported values of 0.2 μM on Hb/ZrO 2/[BMIM]BF4/CHI/GCE [44] and 0.63 μM on Hb/PI/COOH– MWCNTs/GCE [45]. The value of KMapp was further calculated as 10.49 μM, which was lower than the reported values of 7.48 mM on TiO 2 sol–gel/Hb–CSNs/GCE [46] and 4.4 mM on Hb– PVA–GNP–OMIMPF 6 /GCE [47]. Based on the references [48,49],
240
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241
Table 1 Comparison of electrochemical parameters of Hb modified electrodes and the analytical performance for H2O2. Hb modified electrodes
Epa (mV)
Epc (mV)
ΔEp (mV)
α
ks (s−1)
Γ⁎ (mol cm−2)
KMapp (μM)
Linear range (μM)
Detection limit (μM)
Ref.
Hb/ZnSnNPs/MWCNTs/GCE Hb–GR–chitosan/GCE Hb/IL/PDDA–G/GCE a GR–Fe3O4–Hb/GCE Hb/GR–ZnO nanosphere/Au Hb/GR–Pt/GCE Nafion–Hb–CoO–IL/CILE Hb/chitosan–[BMIM]PF6–TiO2–GR/GCE Ag@TiO2/GCE Nafion/Hb–GO–IL/CILE
−444 −103 −292 −285 0 −200 −198 −148 / −148
−358 −324 −360 −363 −132 −320 −278 −265 / −221
86 221 68 78 132 120 80 117 / 73
0.48 / / 0.53 0.5 / 0.423 / / 0.57
0.86 / / 0.91 1.0 0.14 1.571 0.73–3.96 / 0.92
3.93 × 10−10 3.1 × 10−10 4.25 × 10−10 / 1.53 × 10−9 4.53 × 10−10 2.47 × 10−9 3.21 × 10−10 / 3.04 × 10−9
379 344 / 3.7 1.46 × 103 540 16 1.245 × 103 / 13.42
0.5–84.00 6.5–230.0 0.2–32.6 1.5–585.0 1.8–2300.0 10.0–1000.0 8.0–140.0 1.0–1170.0 0.83–43.3 0.08–635.0
0.11 0.51 0.4 0.5 0.6 1.0 / 0.3 0.83 0.0137
[35] [36] [37] [38] [39] [40] [41] [42] [43] This work
the possible electrocatalytic reduction scheme can be expressed with the following equations: −
Hb hemeFeðIIIÞ þ e →Hb hemeFeðIIÞ
−
þ
ð10Þ
−
NO2 þ 2H þ 2e →NO þ H2 O
ð11Þ
Hb hemeFeðIIÞ þ NO→Hb hemeFeðIIÞNO
ð12Þ
The electrochemical reduction of Hb hemeFe(III) resulted in Hb hemeFe(II) (Eq. (10)), and NO− 2 can be reduced to NO (Eq. (11)), then the formed NO can be rapidly bound to heme iron of Hb to get Hb hemeFe(II)NO with a irreversible chemical reaction. So the Hb modified electrode can facilitate the reduction of NO− 2 . 3.6. Analytical application The application of this biosensor was evaluated by determining the residual hydrogen peroxide content in a sterilized milk sample, which was purchased from local supermarket. The standard addition method was used to calculate the recovery with the results summarized in Table 2. The samples were also detected by the traditional KMnO4 titration method. No residual H2O2 was found in the milk sample and the results were in agreement with the proposed method. Also the recovery was in the range from 97.3 to 102.9%, showing a satisfactory result.
compounds that maybe present in the biological or environmental samples. With the addition of different kinds of interferents into the buffer solution and the electrochemical responses were further recorded. Then the changes of the peak currents were calculated. Experimental results indicated that the presence of 1.0 mM K+, Zn2+, Ni2+, Mg2+, Cd2+, Cl−, SO2− 4 and 0.1 mM of ascorbic acid, uric acid, cytosine etc. had less influence with the electrochemical responses changed less than ±5%. So the modified electrode exhibited good selectivity for the electrochemical determination. 4. Conclusions A GO–IL nanocomposite was prepared and used for the realization of the direct electrochemistry of Hb on a HPPF6 based CILE. Due to the specific properties of GO, IL and the composite, direct electron transfer of Hb was achieved with fast electron transfer rate. The modified electrode exhibited good electrocatalytic activity toward the reduction of TCA, H2O2 and NaNO2 with wider linear range and lower detection limit. Therefore this approach provided a new strategy for the development of the third-generation electrochemical biosensors with the advantages including good stability, high electrocatalytic ability and simply preparation procedure. Acknowledgment We acknowledge the financial support provide by the National Natural Science Foundation of China (21075071, 21365010), the Natural Science Foundation of Hainan Province (213015) and the Foundation of Hainan Normal University.
3.7. Reproducibility, stability and selectivity References The reproducibility, stability and selectivity of Nafion/Hb–GO–IL/CILE were investigated. Five independent determinations to a 20.0 mM TCA solution showed a relative standard deviation (RSD) of 2.19%, indicating the modified electrode possessed an acceptable reproducibility. To examine the stability, the response of the Nafion/Hb–GO–IL/CILE was examined with respect to the storage time when not in use. The current value decreased for 3.8% and 5.4% of the initial response after 14 days and 30 days storage, respectively, demonstrating the excellent stability of this biosensor. Thus, Nafion/Hb–GO–IL/CILE exhibited good stability for the electrochemical detection in general. The selectivity of Nafion/ Hb–GO–IL/CILE was evaluated by examining the influence of some
Table 2 Detection results of H2O2 in sterilized milk sample (n = 3). Samples
Detected
Added (μM)
Found (μM)
RSD (%)
Recovery (%)
1 2 3 4
0 0 0 0
10.0 30.0 60.0 90.0
9.73 29.6 61.8 88.3
2.16 1.86 3.13 2.39
97.3 98.5 102.9 98.2
[1] F.A. Armstrong, H.A.O. Hill, N.J. Walton, Acc. Chem. Res. 21 (1988) 407–413. [2] J.F. Rusling, Acc. Chem. Res. 31 (1998) 363–369. [3] N.O. Weiss, H.L. Zhou, L. Liao, Y. Liu, S. Jiang, Y. Huang, X.F. Duan, Adv. Mater. 24 (2012) 5782–5825. [4] V. Singh, D. Joung, L. Zhai, S. Das, S.I. Khondaker, S. Seal, Prog. Mater. Sci. 56 (2011) 1178–1271. [5] D. Chen, H.B. Feng, J.H. Li, Chem. Rev. 112 (2012) 6027–6053. [6] D.R. Dreyer, S.J. Park, C.W. Bielawski, R.S. Ruoff, Chem. Soc. Rev. 39 (2010) 228–240. [7] Y. Liu, D.S. Yu, C. Zeng, Z.C. Miao, L.M. Dai, Langmuir 26 (2010) 6158–6160. [8] J.A. Laszlo, D.L. Compton, J. Mol. Catal. B Enzym. 18 (2002) 109–120. [9] W. Sun, R.F. Gao, K. Jiao, Chin. J. Anal. Chem. 35 (2007) 1813–1819. [10] J.A.M. Shiddiky, A.A.J. Torriero, Biosens. Bioelectron. 26 (2011) 1775–1787. [11] N. Maleki, A. Safavi, F. Tajabadi, Anal. Chem. 78 (2006) 3820–3826. [12] R.T. Kachoosangi, M.M. Musameh, I.A. Yousef, J.M. Yousef, S.M. Kanan, L. Xiao, S.G. Davies, A. Russell, R.G. Compton, Anal. Chem. 81 (2009) 435–442. [13] W. Sun, C.X. Guo, Z.H. Zhu, C.M. Li, Electrochem. Commun. 11 (2009) 2105–2108. [14] K.J. Huang, J.Y. Sun, D.J. Niu, W.Z. Xie, W. Wang, Colloid Surf. B 78 (2010) 69–74. [15] C.X. Ruan, T.T. Li, Q.J. Niu, M. Lu, J. Lou, W.M. Gao, W. Sun, Electrochim. Acta 64 (2012) 183–189. [16] R. Li, C.X. Liu, M. Ma, Z.G. Wang, G.Q. Zhan, B.H. Li, X. Wang, H.F. Fang, H.J. Zhang, C.Y. Li, Electrochim. Acta 95 (2013) 71–79. [17] W.S. Hummers, R.E. Offeman, J. Am. Chem. Soc. 80 (1958) 1339–1340. [18] T.R. Zhan, Y.Q. Guo, L. Xu, W.L. Zhang, W. Sun, W.G. Hou, Talanta 94 (2012) 189–194. [19] P. George, C. Hanania, Biochem. J. 55 (1953) 236–243. [20] R.S. Nicholson, Anal. Chem. 37 (1965) 1351–1352.
W. Sun et al. / Materials Science and Engineering C 40 (2014) 235–241 [21] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, Wiley, New York, 1980. [22] S.F. Wang, T. Chen, Z.L. Zhang, X.C. Shen, Z.X. Lu, D.W. Pang, K.Y. Wong, Langmuir 21 (2005) 9260–9266. [23] E. Laviron, J. Electroanal. Chem. 52 (1974) 355–359. [24] E. Laviron, J. Electroanal. Chem. 101 (1979) 19–28. [25] Y.Q. Wang, H.J. Zhang, D. Yao, J.J. Pu, Y. Zhang, X.R. Gao, Y.M. Sun, J. Solid State Electrochem. 17 (2013) 881–887. [26] W. Wei, H.H. Jin, G.C. Zhao, Microchim. Acta 164 (2009) 167–171. [27] A.A. Ordaz, F. Bedioui, Sens. Actuators B 59 (1999) 128–133. [28] X. Ma, X.J. Liu, H. Xiao, G.X. Li, Biosens. Bioelectron. 20 (2005) 1836–1842. [29] T.R. Zhan, M.Y. Xi, Y. Wang, W. Sun, W.G. Hou, J. Colloid Interface Sci. 346 (2010) 188–193. [30] W. Sun, Y.Q. Guo, Y.P. Lu, A.H. Hu, F. Shi, T.T. Li, Z.F. Sun, Electrochim. Acta 91 (2013) 130–136. [31] R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198–1205. [32] C.X. Ruan, T.T. Li, X.M. Ju, H.J. Liu, J. Lou, W.M. Gao, W. Sun, J. Solid State Electrochem. 16 (2012) 3661–3666. [33] L.L. Zhang, H.H. Cheng, H.M. Zhang, L.T. Qu, Electrochim. Acta 65 (2012) 122–126. [34] Q. Zhang, S.J. Yang, J. Zhang, L. Zhang, P.L. Kang, J.H. Li, J.W. Xu, H. Zhou, X.M. Song, Nanotechnology 22 (2011) 494010–494016. [35] A.L. Sun, H.Y. Zhao, J.B. Zheng, Talanta 88 (2012) 259–264. [36] H.F. Xu, H. Dai, G.N. Chen, Talanta 81 (2010) 334–338. [37] K.P. Liu, J.J. Zhang, G.H. Yang, C.M. Wang, J.J. Zhu, Electrochem. Commun. 12 (2010) 402–405. [38] Y.P. He, Q.L. Sheng, J.B. Zhen, M.Z. Wang, B. Liu, Electrochim. Acta 56 (2011) 2471–2476. [39] J. Xu, C.H. Liu, Z.F. Wu, Microchim. Acta 172 (2011) 425–430. [40] X.M. Feng, R. Li, C.H. Hu, W.H. Hou, J. Electroanal. Chem. 657 (2011) 28–33. [41] Z.H. Zhu, X. Li, Y. Zeng, W. Sun, W.M. Zhu, X.T. Huang, J. Phys. Chem. C 115 (2011) 12547–12553. [42] J.Y. Sun, K.J. Huang, S.F. Zhao, Y. Fan, Z.W. Wu, Bioelectrochemistry 82 (2011) 125–130.
[43] [44] [45] [46] [47] [48] [49]
241
M.M. Khan, S.A. Ansari, J. Lee, M.H. Cho, Mater. Sci. Eng. C 33 (2013) 4692–4699. Y.H. Ma, G.Q. Zhan, M. Ma, X. Wang, C.Y. Li, Bioelectrochemistry 84 (2012) 6–10. H.H. Kou, L.P. Jia, C.M. Wang, W.C. Ye, Electroanalysis 24 (2012) 1799–1803. S. Zhao, K. Zhang, Y.Y. Sun, C.Q. Sun, Bioelectrochemistry 69 (2006) 10–15. Y.F. Zhang, R. Yan, F.Q. Zhao, B.Z. Zeng, Colloid Surf. B 71 (2009) 288–292. J.N. Younathan, K.S. Wood, T.J. Meyer, Inorg. Chem. 31 (1992) 3280–3285. W.W. Yang, Y. Bai, Y.C. Li, C.Q. Sun, Anal. Bioanal. Chem. 382 (2005) 44–50.
Wei Sun is a professor in the College of Chemistry and Chemical Engineering, Hainan Normal University. He received his PhD in analytical chemistry from Ocean University of China in 2002. His current interests are bioelectroanalysis. Shixing Gong is a master candidate in the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology. Fan Shi is a master candidate in the College of Chemistry and Chemical Engineering, Hainan Normal University. Lili Cao is a master candidate in the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology. Luyang Ling is an undergraduate student in the College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology. Weizhe Zheng is an undergraduate student in the College of Chemistry and Chemical Engineering, Hainan Normal University. Wencheng Wang is a master candidate in the College of Chemistry and Chemical Engineering, Hainan Normal University.
