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Electrochemistry and electrocatalysis of hemoglobin on multi-walled carbon nanotubes modified carbon ionic liquid electrode with hydrophilic EMIMBF4 as modifier
Electrochimica Acta 54 (2009) 4141–4148
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Electrochemistry and electrocatalysis of hemoglobin on multi-walled carbon nanotubes modified carbon ionic liquid electrode with hydrophilic EMIMBF4 as modifier Wei Sun ∗ , Xiaoqing Li, Yan Wang, Ruijun Zhao, Kui Jiao Key Laboratory of Eco-Chemical Engineering of Ministry of Education, 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 22 December 2008 Received in revised form 10 February 2009 Accepted 17 February 2009 Available online 26 February 2009 Keywords: Hemoglobin Multi-walled carbon nanotubes Carbon ionic liquid electrode 1-Ethyl-3-methylimidazolium tetrafluoroborate Direct electron transfer
a b s t r a c t The direct electrochemistry of hemoglobin (Hb) on multi-walled carbon nanotubes (MWCNTs) modified carbon ionic liquid electrode (CILE) was achieved in this paper. By using a hydrophilic ionic liquid 1ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 ) as the modifier, a new CILE was fabricated and further modified with MWCNTs to get the MWCNTs/CILE. Hb molecules were immobilized on the surface of MWCNTs/CILE with polyvinyl alcohol (PVA) film by a step-by-step method and the modified electrode was denoted as PVA/Hb/MWCNTs/CILE. UV–vis and FT-IR spectra indicated that Hb remained its native structure in the composite film. Cyclic voltammogram of PVA/Hb/MWCNTs/CILE showed a pair of well defined and quasi-reversible redox peaks with the formal potential (E0 ) of −0.370 V (vs. SCE) in 0.1 mol/L pH 7.0 phosphate buffer solution (PBS), which was the characteristic of the Hb heme FeIII /FeII redox couples. The redox peak currents increased linearly with the scan rate, indicating the direct electron transfer was a surface-controlled process. The electrochemical parameters of Hb in the film were calculated with the results of the electron transfer coefficient (˛) and the apparent heterogeneous electron transfer rate constant (ks ) as 0.49 and 1.054 s−1 , respectively. The immobilized Hb in the PVA/MWCNTs composite film modified CILE showed excellent electrocatalytic activity to the reduction of trichloroacetic acid (TCA) and hydrogen peroxide. So the proposed electrode showed the potential application in the third generation reagentless biosensor. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The direct electrochemistry of redox proteins has aroused great interests in recent years for the importance in realizing the mechanisms of electron transfer in real biological systems and constructing third-generation mediatorless biosensors. However, the direct electron transfer (DET) between the redox proteins and the traditional bare working electrode is difficult to be realized due to the deep burying of the electroactive prosthetic groups within the protein structure [1]. Therefore different kinds of films modified electrodes including surfactants [2,3], hydrogel polymers [4,5], nanoparticles [6–8] and biomembranes [9,10] are devised to provide a suitable microenvironment for redox proteins to take place the direct electron transfer between the active center of redox proteins and the basal electrode. Different kinds of redox proteins such as hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase
∗ Corresponding author. Tel.: +86 532 84022681; fax: +86 532 84023927. E-mail address: [email protected] (W. Sun). 0013-4686/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2009.02.055
(HRP), Cytochrome c (Cyt c) and glucose oxidase (GOD) have been carefully investigated. Recently Lojou et al. [11] investigated the efficient hydrogenase orientation for H2 oxidation at carbon nanotubes modified electrodes. Due to the advantages of Hb such as commercial availability, moderate cost and well-documented structure, Hb is generally used as a model for the DET process between redox proteins and the modified electrode in the biological systems. In recent years nanomaterials have been widely used in the electrochemical biosensors. Among the nanomaterials used for protein film electrochemistry, carbon nanotubes (CNTs) had been extensively studied after its discovery by Iijima [12]. Due to the specific characteristics such as good electronic and mechanical properties, electric conductivity and biocompatibility, CNTs modified electrodes had been applied in the field of electroanalysis [13]. By incorporating CNTs on the surface of modified electrode, the electron transfer rate of electroactive substances are greatly improved. In the protein electrochemistry the presence of CNTs can provide an effective electron-conducting tunnel, reduce the insulating property of proteins, facilitate the electron transfer between electrode and redox protein. For example, Liu et al. reported direct electron transfer of Hb on the cetyltrimethylammonium bromide and
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CNTs modified electrode [14]. Luo et al. reported an electrodeposited nanocomposite of chitosan and CNTs modified electrode for biosensor [15]. Carbon nanotubes can be combined with other materials such as Teflon [16], Nafion [17,18], Nafion and platinum nanoparticles composite [19], sol–gel-derived ceramics [20], etc. to get different modified electrodes for protein electrochemistry. Recently room temperature ionic liquids (RTILs) have attracted great attentions in the field of chemistry due to their excellent properties such as negligible vapor pressure, high chemical and thermal stability, good conductivity and dissolving capability. The reviews about the application of RTILs in the fields of analytical chemistry or electrochemistry had been reported [21,22]. Due to the advantages such as higher ionic conductivity and wider electrochemical windows, RTILs can be used as the modifier or the supporting electrolyte in the field of electroanalysis. Wei and Ivaska reviewed the recent progress of RTILs in the electrochemical sensors [23]. Compared with other modified electrodes, ionic liquid modified carbon paste electrode (CILE) shows some advantages such as higher sensitivity, wider electrochemical windows and good anti-fouling ability [24,25]. So CILE had been used as the basal electrode for the investigation on the redox proteins electrochemistry or applied to the detection of some electroactive substances [26–30]. Wang et al. also applied the RTILs as the electrolyte for the investigation of the direct electrochemistry of heme proteins [31,32]. In this paper, a CILE was first fabricated by using a hydrophilic ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 ) as modifier and further modified with multiwalled carbon nanotubes (MWCNTs). Hemoglobin was immobilized on the surface of MWCNTs/CILE and subsequently fixed by a layer of polyvinyl alcohol (PVA) film. PVA is a polymer composed of a carbon chain backbone attached with hydroxyl groups, which can provide a biocompatible microenvironment for proteins or enzyme immobilization and improve the stability of the modified electrode. The modified electrode was characterized by different methods such as scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The direct electron transfer of Hb with the modified electrode was achieved and the results indicated that the MWCNTs/CILE could effectively facilitate the direct electrochemistry of Hb. The electrocatalysis of the resulting electrode to the reduction of hydrogen peroxide (H2 O2 ) and trichloroacetic acid (TCA) were further studied. 2. Experimental 2.1. Reagents Ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 , Hangzhou Kemer Chemical Limited Company), bovine hemoglobin (Hb, MW. 64500, Tianjin Chuanye Biochemical Limited Company), multi-walled carbon nanotubes (MWCNTs, purity >95%, main range of diameter <10 nm, length of 5–15 m, Shenzhen Nanoport Company, China), polyvinyl alcohol (PVA, average degree of polymerization 7500, MW. 14,000, Tianjin Bode Chemical Limited Company), graphite powder (average particle size 30 m, Shanghai Colloid Chemical Company), trichloroacetic acid (TCA, Tianjin Kemiou Chemical Limited Company) were used as received. 0.1 mol/L 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 all the experiments. 2.2. Apparatus A CHI 750B electrochemical workstation (Shanghai CH Instrument, China) was used for all the electrochemical measurements
including cyclic voltammetry and electrochemical impedance spectroscopy. A conventional three-electrode system was used with a Hb modified electrode as working electrode, a platinum wire as auxiliary electrode and a saturated calomel electrode (SCE) as reference electrode. UV–vis absorption spectra and FT-IR spectra were recorded on Cary 50 probe spectrophotometer (Varian Company, Australia) and Tensor 27 FT-IR spectrophotometer (Bruker Company, Germany), respectively. Scanning electron microscopy was performed on a JSM-6700F scanning electron microscope (Japan Electron Company, Japan). 2.3. Preparation of PVA/Hb/MWCNTs/CILE CILE was fabricated with the following procedure: 0.15 mL of EMIMBF4 , 0.85 mL of liquid paraffin and 3.2 g of graphite powder were hand-mixed in an agate mortar and ground carefully. A portion of resulting homogeneous paste was packed firmly into a glass tube cavity (˚ = 4 mm). The electric contact was established through a copper wire to the end of the paste in the inner hole of the tube and the surface of CILE was polished by smoothing on a weighing paper. The traditional carbon paste electrode was prepared by mixing graphite powder with liquid paraffin at a ratio of 70/30 (w/w) according to the general procedure. The MWCNTs modified CILE (MWCNTs/CILE) was prepared by simply applying 3.0 L of 1.0 g/L MWCNTs suspension solution (dispersed in dimethyl formamide, DMF) on the surface of CILE and left it to dry at the room temperature for 2 h. Then 10.0 L of 20.0 mg/mL Hb solution was dropped on the surface of MWCNTs/CILE and stayed silent to allow the water evaporated gradually. At last 10.0 L of 2.0 mg/mL PVA solution was cast on the surface of electrode to form a stable film. During these procedures a small bottle was fit over the electrode so that the solvent could evaporate slowly to get a uniform film. The resulted modified electrode was denoted as PVA/Hb/MWCNTs/CILE and stored at 4 ◦ C when not in use. Other modified electrodes including PVA/hemin/MWCNTs/CILE, PVA/Hb/MWCNTs/CPE, PVA/MWCNTs/CILE, etc. were prepared by the similar procedures and used in the experiment for comparison. 2.4. Procedure The electrochemical measurements were carried out in a 10-mL electrochemical cell containing 0.1 mol/L PBS, which was purged with highly purified nitrogen for 30 min prior to a series of experiments and maintained in a nitrogen atmosphere during the experiments. UV–vis spectroscopic experiments were performed with a mixture solution of certain concentration of Hb, MWCNTs and PVA with different pH PBS. The PVA/Hb/MWCNTs film assembled on a glass slide was used for FT-IR measurements. 3. Results and discussion 3.1. Spectroscopic characterizations In UV–vis absorption spectrum the Soret absorption band from the four iron heme groups of heme proteins may provide the information on the conformational integrity of the proteins and the possible denaturation or the conformational change of heme region [33]. As shown in Fig. 1, Hb had a Soret band at 406.0 nm in water (curve a). After mixing Hb, PVA and MWCNTs with different external pH buffer solutions as 5, 7 and 10, respectively, the absorption value also appeared at 406.0 nm (curve b–d). Obviously, these absorption peaks were attributed to the Soret band of Hb and the results suggested that Hb in the composite film retained its native structure in a wide range of buffer pH. While in a pH 2.0 buffer solution, the
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Fig. 1. UV–vis absorption spectra of Hb in water (a) and PVA/Hb/MWCNTs with pH (b) 5.0 (c) 7.0, (d) 10.0, and (e) 2.0 PBS, respectively.
Soret band became broader and smaller with the maximum absorption band at 368.0 nm (curve e), indicating that Hb molecules were denatured to a certain extent in the acidic solution. FT-IR spectroscopy is also a sensitive method to probe into the secondary structure of proteins. The profiles of the amide I and amide II infrared absorbance bands of Hb provide detailed information on the secondary structure of the polypeptide chain. The amide I band at 1700–1600 cm−1 is attributed to the C O stretching vibra-
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Fig. 2. FT-IR spectra of (a) Hb and (b) PVA/Hb/MWCNTs film.
tion of the peptide linkage in the backbone of protein. The amide II band at 1600–1500 cm−1 is caused by the combination of N H inplane bending and C N stretching vibration of the peptide groups [34]. If Hb molecule is denatured, the intensity and shape of the amide I and amide II bands will diminish or even disappear [35,36]. As shown in Fig. 2, the FT-IR spectra of amide I and II bands of native Hb were located at 1655.34 and 1535.80 cm−1 , respectively (Fig. 2a). After mixing Hb, PVA and MWCNTs together, the spectra of amide I and amide II bands appeared at 1653.55 and 1538.66 cm−1 (Fig. 2b).
Fig. 3. SEM images of (a) CILE, (b) MWCNTs/CILE, (c) Hb/MWCNTs/CILE and (d) PVA/Hb/MWCNTs/CILE.
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The similarities of the two spectra suggested that Hb retained the essential features of its native structure after immobilized in the PVA and MWCNTs composite films. 3.2. Surface morphologies of modified electrode Scanning electron microscopy was used to characterize the top views of the different modified electrodes and the results were shown in Fig. 3. On CILE (Fig. 3a) an uniform surface appeared without separated carbon layer, which was due to the embedment of ionic liquids EMIMBF4 between the layer of carbon and disperse the carbon powder homogeneously. While on the MWCNTs/CILE (Fig. 3b), there were plenty of tubes dispersed on the surface, which indicated that MWCNTs were successfully immobilized on the surface of CILE. After MWCNTs/CILE was further coated with Hb molecules, the aggregation of the immobilized Hb molecules was distributed regularly and showed a network-like structure (Fig. 3c). On the PVA/Hb/MWCNTs/CILE (Fig. 3d) an unshaped membrane appeared, which was due to the presence of PVA film on the electrode surface. 3.3. Characteristics of the modified electrodes Electrochemical impedance spectroscopy can provide information on the impedance changes of the electrode surface during the modification process. By using [Fe(CN)6 ]3−/4− redox couples as the electrochemical probe, the Nyquist plots of different modified electrodes were shown in Fig. 4. On the bare CILE, the electron transfer resistance (Ret), which was derived from the semicircle domains of impedance spectra, can be estimated to be 667.4 (curve a). After PVA was coated on the surface of CILE, the Ret value was increased to 1102.3 (curve b), which indicated a film of polymer PVA formed on the surface could hinder the electron transfer. While on MWCNTs/CILE, the Ret value was decreased dramatically and nearly to zero (curve c), indicating that MWCNTs present on the surface of CILE could enhance the conductivity. Then the Ret value increased to 83.7 after the PVA was coated on the MWCNTs/CILE (curve d). Subsequently, when Hb was immobilized on the PVA/MWCNTs/CILE the Ret value increased again (curve e), which indicated that Hb molecules were successfully immobilized in the PVA/MWCNTs films. Cyclic voltammetric results of ferricyanide with different modified electrodes are also a valuable and convenient tool to
Fig. 4. EIS for (a) bare CILE, (b) PVA/CILE, (c) MWCNTs/CILE, (d) PVA/MWCNTs/CILE and (e) PVA/Hb/MWCNTs/CILE in a mixture solution of 10.0 mmol/L [Fe(CN)6 ]3−/4− and 0.1 mol/L KCl with the frequencies swept from 105 to 0.1 Hz under a open circuit potential conditions.
Fig. 5. Cyclic voltammograms of (a) bare CILE, (b) PVA/CILE, (c) MWCNTs/CILE, (d) PVA/MWCNTs/CILE and (e) PVA/Hb/MWCNTs/CILE in a mixture solution of 1.0 mmol/L [Fe(CN)6 ]3− and 0.1 mol/L KCl with the scan rate as 100 mV s−1 .
monitor the modification process on the modified electrodes. Cyclic voltammograms of different modified electrodes with 1.0 mmol/L [Fe(CN)6 ]3− solution were recorded with the results shown in Fig. 5. A pair of well-defined redox peaks was observed at the bare CILE (curve a). When PVA was coated on the bare CILE, the redox peak currents decreased drastically (curve b), implying the presence of PVA film on the electrode surface acted as an inert layer and blocked the diffusion of ferricyanide toward the electrode. However, on the MWCNTs/CILE, the electrochemical response was bigger than that of bare CILE (curve c), which indicated that the presence of MWCNTs could effectively improve the conductivity of the electrode. When PVA was coated on the MWCNTs/CILE, the redox peak current decreased correspondingly (curve d). After Hb was further immobilized in the PVA/MWCNTs/CILE, the redox peak current was further decreased (curve e), which indicated that Hb molecules in the film further blocked the diffusion of ferricyanide. The data were in consistent with the results obtained from EIS experiments. On the basis of the EIS and cyclic voltammetric results, we can conclude that Hb was successfully immobilized in the PVA/MWCNTs composite films on the surface of CILE. 3.4. Direct electrochemistry of Hb Fig. 6 shows the typical cyclic voltammograms of different electrodes in pH 7.0 PBS at the scan rate of 200 mV s−1 . No electrochemical responses were observed at bare CILE (curve a), PVA/CILE (curve b) and MWCNTs/CILE (curve c), which indicated no electroactive substances existed on the electrode surface. While on the PVA/Hb/CILE a pair of small and unsymmetric redox peaks appeared (curve d), which indicated that the direct electron transfer of Hb with CILE had taken place with slow electron transfer rate, and the quasi-reversible electrochemical responses appeared. Since CILE had the advantages of high conductivity, high sensitivity, good anti-fouling ability and a layer of IL was presented on the surface of CILE [24,25], it can provide a suitable interface to slightly promote the electron transfer reaction of protein. While on the PVA/Hb/MWCNTs/CILE, the electrochemical response of Hb was greatly enhanced and a pair of well-defined quasi-reversible cyclic voltammetric peaks was observed (curve f). For comparison the cyclic voltammogram of PVA/Hb/MWCNTs/CPE was also recorded (curve e). Only a small reduction peak appeared without the oxidation peak, indicating that the direct electron transfer process was not realized on the MWCNTs modified CPE. Although MWCNTs have many specific advantages such as large surface area,
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trode for the reduction or oxidation of electroactive species in the thin films. According to the Q– * relationship equation: Q = nFA * [41], the surface concentration ( *) of electroactive Hb in the film was calculated with the average value as 3.5 × 10−9 mol cm−2 . The results were larger than the theoretical monolayer value (1.89 × 10−11 mol cm−2 ) of protein [31], indicating that several layers of protein could exchange electrons with the electrode, which may be due to the presence of MWCNTs on the electrode surface resulted in a three-dimensional structure for Hb immobilization. The total amount of Hb in the modified film was calculated as 2.47 × 10−8 mol cm−2 based on the volume and the concentration of Hb solution deposited on the electrode surface. So the fraction of electroactive Hb was calculated as 14.2%. With the increase of the scan rate the peak-to-peak separation (Ep) were also increased gradually, so the electrochemical parameters could be calculated using the method developed by Laviron [42,43], which was valid for flat film modified electrode with surface-controlled electrochemical process at the value of Ep < 200 mV: Fig. 6. Cyclic voltammograms of (a) bare CILE, (b) PVA/CILE, (c) MWCNTs/CILE, (d) PVA/Hb/CILE, (e) PVA/Hb/MWCNTs/CPE and (f) PVA/Hb/MWCNTs/CILE in pH 7.0 PBS at the scan rate of 200 mV s−1 . Inset: cyclic voltammogram of PVA/hemin/MWCNTs/CILE in pH 7.0 PBS at the scan rate of 200 mV s−1 .
Epc = E 0 −
high biocompatible interface and good electric conductivity, the presence of CILE also played important roles in realizing the direct electron transfer process. So the synergistic effect of CILE and MWCNTs played important roles in achieving the redox reaction of Hb in the composite film. As indicated in references [37–39], a layer of IL existed on the surface of CILE and MWCNTs could interact with the imidazolium group on the electrode surface through – or/and -cationic, hydrophobic and electrostatic interaction. Then a stable MWCNT–IL composite film was formed on the surface of electrode for the further Hb immobilization. From the cyclic voltammogram the redox peak potentials were got with the cathodic peak potential (Epc) as −0.436 V and the anodic peak potential (Epa) as −0.303 V (vs. SCE). The formal potential (E0 ), 0 which is calculated from the equation as E = (Epa + Epc)/2, was estimated as −0.370 V (vs. SCE) and it was the characteristic of heme FeIII /FeII redox couples [40]. The peak-to-peak separation (Ep) at 200 mV s−1 was got as 133 mV and the ratio of redox peak current (Ipa/Ipc) was approximately to be 1. The results indicated a quasi-reversible electrochemical behavior. Also the cyclic voltammogram of PVA/hemin/MWCNTs/CILE was further investigated (insert of Fig. 6). A pair of small and unsymmetric redox peaks with Epc as −0.547 V and Epa as −0.348 V (vs. SCE) appeared. The formal potential (E0 ) was estimated as −0.448 V (vs. SCE). The results were different with that of PVA/Hb/MWCNTs/CILE, indicating that the Hb molecules in the composite film was not denatured or degenerated. Cyclic voltammograms of PVA/Hb/MWCNTs/CILE with different scan rates from 50 to 800 mV s−1 in pH 7.0 PBS were further recorded. A pair of quasi-reversible redox peaks appeared with the almost equal high of the redox peak current, which indicated that all the electroactive heme FeIII of the protein in the film was reduced to heme FeII in the forward cathodic scan with the complete conversion of heme FeII back to its FeIII form in the reversed anodic scan. Both the redox peak currents increased with the scan rate and two linear regression equations were got as Ipc (A) = 0.0054 (V s−1 ) + 0.558 with a correlation coefficient () of 0.999 and Ipa (A) = −0.0058 (V s−1 ) −0.326 ( = 0.995), which indicated a typical characteristic of diffusionless surface-controlled thin-layer electrochemical behavior. For a thin-layer electrochemistry, the integration of cyclic voltammetric peaks of protein film electrode in the buffer gives the total amount of charge value (Q) passed through the elec-
Epa = E 0 +
RT ln ˛nF
(1)
RT ln (1 − ˛)nF
(2)
log ks = ˛ log (1 − ˛) + (1 − ˛) log ˛ − log nFEp − ˛(1 − ˛) 2.3RT
2.3RT nF (3)
where ˛ is the electron transfer coefficient, n is the electron transfer number, ks is the apparent heterogeneous electron transfer rate constant, R is the gas constant, T is the absolute temperature, and Ep is the peak-to-peak separation. The relationship of Epc and Epa with ln were constructed with the linear regression equations as Epa (V) = 0.0216 ln − 0.268 ( = 0.996) and Epc (V) = −0.041 ln − 0.502 ( = 0.994). According to Eqs. (1) and (2) the electron transfer coefficient (˛) was calculated as 0.49. Based on Eq. (3) the apparent heterogeneous electron transfer rate constant (ks ) was further calculated from the relationship of Ep with ln and the result was got as 1.054 s−1 , which was higher than some previous reports with casting method for protein immobilization [44–46], indicating that the PVA/MWCNTs/CILE provided a suitable microenvironment for the increase of electron transfer rate of Hb. But the value was smaller than most of the Hb biosensor fabricating by layer-by-layer method, which may be due to the different preparation methods provide different microenviroment for Hb molecules to transfer electron [47].
3.5. Effect of buffer pH The direct electrochemistry of the Hb immobilized in PVA/MWCNTs film showed a strong dependence on pH value of external solutions with the cyclic voltammograms shown in Fig. 7. An increase of buffer pH led to the negative shift of both the reduction and oxidation peak potentials. The formal potential (E0 ) had a linear relationship with pH value from 4.0 to 10.0 with the linear regression equation as E0 (V) = −0.0422 pH −0.0305 ( = 0.998). The value of slope was −42.2 mV/pH, which was reasonably smaller to the theoretical value of −56.0 mV/pH at 20 ◦ C for the reversible one-electron transfer coupled with single-proton transportation. But the electrode process can also be represented as Hb FeIII + H+ + e Hb FeII , where the charges of Hb molecules are omitted [48].
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Fig. 7. Influence of pH on cyclic voltammograms of PVA/Hb/MWCNTs/CILE with 4.0, 6.0, 8.0, and 10.0 of the pH values, respectively, at the scan rate of 200 mV s−1 .
3.6. Electrocatalytic reactivity The PVA/Hb/MWCNTs/CILE showed good electrocatalytic ability to different substrates such as H2 O2 and trichloroacetic acid. As shown in Fig. 8, with the addition of H2 O2 into the pH 7.0 PBS an obvious increase of the cathodic peak current at −0.436 V (vs. SCE) appeared with the decrease of the anodic peak current (curves a–f). However no similar phenomena could be observed at other modified electrodes without the Hb under the same conditions. The more the H2 O2 added, the larger the cathodic peak current got, which indicated the Hb incorporated in the composite film could act as an effective catalyst to the reduction of H2 O2 . A linear relationship between the catalytic peak current and H2 O2 concentration was obtained (inset of Fig. 8) in the range from 1.2 to 30.0 mol/L with the linear regression equation as Ip (A) = 0.286 C (mol/L) + 0.256 ( = 0.997) and the detection limit as 1.0 mol/L (3). The relative standard deviation (R.S.D.) for the six successive determinations of 4.0 mol/L H2 O2 was calculated as 3.7%. When the concentration of H2 O2 was more than 50.0 mol/L, a plateau of catalytic peak current appeared, which suggested a typical electrocatalytic process with Michaelis–Menten kinetic model. The apparent Michaelis–Menten app constant (KM ) was further calculated from the electrochemical
Fig. 9. Cyclic voltammograms of PVA/Hb/MWCNTs/CILE in 0.1 mol/L pH 7.0 PBS containing 0, 0.8, 3.6, 4.4, 6.0, 8.0, and 10.0 mmol/L TCA (curves a–h), respectively, with the scan rate of 100 mV s−1 .
version of the Lineweaver–Burk equation [49], which could provide an indicative of the enzyme–substrate kinetics. app
K 1 1 = + M Iss Imax Imax c
(4)
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 the saturated substrate condition. By an analysis the slope and the intercept of the plot of the reciprocals of the reduction peak current versus H2 O2 concentration, the app KM value was calculated as 0.928 mmol/L, which was smaller than app some reported H2 O2 biosensors [50,51]. The smaller KM value indicated that the higher activity of immobilized Hb in the composite film. Electrocatalytic activity of PVA/Hb/MWCNTs/CILE towards TCA was also investigated with the results shown in Fig. 9. When TCA was added into a pH 7.0 PBS, an increase in the Hb FeIII reduction peak current was observed with the decrease of Hb FeII oxidation peak. This may be due to the reaction of Hb FeII with TCA to produce Hb FeIII again, which then reduced electrochemically at electrode to form a catalytic cycle. The catalytic reduction peak current increased with the TCA concentration in the range from 0.5 to 12.0 mmol/L and the linear regression equation was got as Ip (mA) = 0.0322 C (mmol/L) + 0.0015 ( = 0.998) with the detection app limit as 0.1 mmol/L (3). According to Eq. (4) the KM value of the enzyme–substrate kinetic reaction was calculated as 5.13 mmol/L, which was lower than that of the previous report [48]. 3.7. Stability and repeatability of the modified electrode
Fig. 8. Cyclic voltammograms of PVA/Hb/MWCNTs/CILE in pH 7.0 PBS containing 0, 1.0, 3.0, 4.0, 5.0, and 20.0 mol/L H2 O2 (from a–f), respectively, at the scan rate of 100 mV s−1 . Inset: the relationship between the peak current and the concentration of H2 O2 .
The stability of the CILE was first investigated. Since EMIMBF4 was a hydrophilic ionic liquid which is totally water miscible, it was used as modifier in the carbon paste electrode. The ratio of liquid paraffin and EMIMBF4 was optimized, which showed great influence on the stability of the CILE. The peak current remained stable in the ratio range from 9/1 to 4/1. When the ratio was more than 7/3, the peak current tended to be unstable due to the partly dissolution of EMIMBF4 into the solution. So the final volume ratio of liquid paraffin and EMIMBF4 was selected as 85/15, which gave stable response for 100 continuous scanning in pH 7.0 PBS. The storage of the CILE also showed good stability in 1 month without the changes of the background currents in the same buffer solution.
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Table 1 Comparison of different redox protein film electrodes for H2 O2 detection. Modified electrode Hb/IL/CILE Hb–Nafion–Co3 O4 –GCE SA/Hb/CILE HRP-PEG/PGE HRP-Chi-BMIMBF4 /GCE Hb-CILE PVA/Hb/MWCNTs/CILE
E0 (V)
* (mol cm−2 )
−0.22 −0.350 −0.344 −0.379 −0.34 −0.334 −0.370
8.4 × 10−11
3.1 × 10−9 3.2 × 10−10 – 1.03 × 10−10 1.38 × 10−9 3.5 × 10−9
Linear range (M) 100–5000 1–390.0 1.0–100.0 2.0–100.0 0.75–135.0 8.0–280.0 1.2–30.0
app
Detection limit (M)
KM (mM)
Reference
40 0.1 1.0 67 0.25 1.0 0.5
7.5 0.136 – 1.38 – 1.103 0.928
[30] [52] [53] [54] [55] [56] This paper
GCE: glassy carbon electrode; SA: sodium alginate; PEG: polyethylene glycol; PGE: pyrolytic graphite electrode; Chi: chitosan; BMIMBF4 : 1-butyl-3-methylimidazolium tetrafluoroborate; MWCNTs: multiwall carbon nanotubes.
Then the stability of PVA/Hb/MWCNTs/CILE was further investigated with two methods. Firstly the modified electrode was evaluated by examining the cyclic voltammetric peak currents after continuous scanning for 50 cycles. There was nearly no decrease of the voltammetric response, indicating that PVA/Hb/MWCNTs/CILE was stable in buffer solution. The storage stability of the Hb modified electrode was investigated by keeping the electrode at 4 ◦ C when the electrode was not in use. 95.3% of the initial current response was retained after 2 weeks storage. After 1 month of testing, the peak current response decreased about 10%. The relatively good stability of the Hb electrode can be attributed to the biocompatibility between the PVA/MWCNTs composite film and Hb. The composite film can prevent the leakage of the proteins and retain electrocatalytic activity of the proteins. Six Hb modified electrodes were made by the same procedure independently and the relative standard deviation of 4.3% was calculated for the determination of 4.0 × 10−6 mol/L H2 O2 , which indicated the modified electrode had good repeatability. 4. Conclusion By using a hydrophilic ionic liquid EMIMBF4 , MWCNTs and PVA as the composite materials, Hb was successfully immobilized on a multi-walled carbon nanotubes modified carbon ionic liquid electrode. The resulting PVA/Hb/MWCNTs/CILE showed good electrochemical behaviors and a pair of well-defined quasi-reversible redox couple of Hb FeIII /FeII was got with the formal potential as −0.370 V (vs. SCE) in pH 7.0 PBS. The results indicated that the synergistic effect of MWCNTs and CILE played important roles in facilitating the direct electron transfer of Hb. The electrochemical parameters of Hb in the film were carefully calculated and the Hb modified electrode showed good electrocatalytic ability to the reduction of H2 O2 and TCA. The comparison of the proposed electrode with other redox protein modified electrodes fabricated with Nafion or other ionic liquids was listed in Table 1. Compared with the another commonly used hydrophobic ionic liquid 1-butyl-3methylimidazolium hexafluorophate (BMIMPF6 ) used for protein app modified electrode, the lower detection limit and small KM value appeared, which may be due to the hydrophilic solvent have strong effect on the activity of the protein. Also EMIMBF4 showed higher conductivity and smaller viscosity than BMIMPF6 . So the electron transfer process was much faster with EMIMBF4 as the modifier. As for the different film forming materials including Nafion and chitosan, it was difficult to evaluate the performance because of different basal electrode or nanomaterials were used. But the high hydrophilic ability of PVA could provide a suitable microenvironment for Hb to keep the bioactivity. So the PVA/Hb/MWCNTs/CILE may have the future application in the third-generation biosensors. Acknowledgements We are grateful to the financial support of the National Science Foundation of China (Nos. 20635020 and 20405008) and the
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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta
Electrochemistry and electrocatalysis of hemoglobin on multi-walled carbon nanotubes modified carbon ionic liquid electrode with hydrophilic EMIMBF4 as modifier Wei Sun ∗ , Xiaoqing Li, Yan Wang, Ruijun Zhao, Kui Jiao Key Laboratory of Eco-Chemical Engineering of Ministry of Education, 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 22 December 2008 Received in revised form 10 February 2009 Accepted 17 February 2009 Available online 26 February 2009 Keywords: Hemoglobin Multi-walled carbon nanotubes Carbon ionic liquid electrode 1-Ethyl-3-methylimidazolium tetrafluoroborate Direct electron transfer
a b s t r a c t The direct electrochemistry of hemoglobin (Hb) on multi-walled carbon nanotubes (MWCNTs) modified carbon ionic liquid electrode (CILE) was achieved in this paper. By using a hydrophilic ionic liquid 1ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 ) as the modifier, a new CILE was fabricated and further modified with MWCNTs to get the MWCNTs/CILE. Hb molecules were immobilized on the surface of MWCNTs/CILE with polyvinyl alcohol (PVA) film by a step-by-step method and the modified electrode was denoted as PVA/Hb/MWCNTs/CILE. UV–vis and FT-IR spectra indicated that Hb remained its native structure in the composite film. Cyclic voltammogram of PVA/Hb/MWCNTs/CILE showed a pair of well defined and quasi-reversible redox peaks with the formal potential (E0 ) of −0.370 V (vs. SCE) in 0.1 mol/L pH 7.0 phosphate buffer solution (PBS), which was the characteristic of the Hb heme FeIII /FeII redox couples. The redox peak currents increased linearly with the scan rate, indicating the direct electron transfer was a surface-controlled process. The electrochemical parameters of Hb in the film were calculated with the results of the electron transfer coefficient (˛) and the apparent heterogeneous electron transfer rate constant (ks ) as 0.49 and 1.054 s−1 , respectively. The immobilized Hb in the PVA/MWCNTs composite film modified CILE showed excellent electrocatalytic activity to the reduction of trichloroacetic acid (TCA) and hydrogen peroxide. So the proposed electrode showed the potential application in the third generation reagentless biosensor. © 2009 Elsevier Ltd. All rights reserved.
1. Introduction The direct electrochemistry of redox proteins has aroused great interests in recent years for the importance in realizing the mechanisms of electron transfer in real biological systems and constructing third-generation mediatorless biosensors. However, the direct electron transfer (DET) between the redox proteins and the traditional bare working electrode is difficult to be realized due to the deep burying of the electroactive prosthetic groups within the protein structure [1]. Therefore different kinds of films modified electrodes including surfactants [2,3], hydrogel polymers [4,5], nanoparticles [6–8] and biomembranes [9,10] are devised to provide a suitable microenvironment for redox proteins to take place the direct electron transfer between the active center of redox proteins and the basal electrode. Different kinds of redox proteins such as hemoglobin (Hb), myoglobin (Mb), horseradish peroxidase
∗ Corresponding author. Tel.: +86 532 84022681; fax: +86 532 84023927. E-mail address: [email protected] (W. Sun). 0013-4686/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.electacta.2009.02.055
(HRP), Cytochrome c (Cyt c) and glucose oxidase (GOD) have been carefully investigated. Recently Lojou et al. [11] investigated the efficient hydrogenase orientation for H2 oxidation at carbon nanotubes modified electrodes. Due to the advantages of Hb such as commercial availability, moderate cost and well-documented structure, Hb is generally used as a model for the DET process between redox proteins and the modified electrode in the biological systems. In recent years nanomaterials have been widely used in the electrochemical biosensors. Among the nanomaterials used for protein film electrochemistry, carbon nanotubes (CNTs) had been extensively studied after its discovery by Iijima [12]. Due to the specific characteristics such as good electronic and mechanical properties, electric conductivity and biocompatibility, CNTs modified electrodes had been applied in the field of electroanalysis [13]. By incorporating CNTs on the surface of modified electrode, the electron transfer rate of electroactive substances are greatly improved. In the protein electrochemistry the presence of CNTs can provide an effective electron-conducting tunnel, reduce the insulating property of proteins, facilitate the electron transfer between electrode and redox protein. For example, Liu et al. reported direct electron transfer of Hb on the cetyltrimethylammonium bromide and
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CNTs modified electrode [14]. Luo et al. reported an electrodeposited nanocomposite of chitosan and CNTs modified electrode for biosensor [15]. Carbon nanotubes can be combined with other materials such as Teflon [16], Nafion [17,18], Nafion and platinum nanoparticles composite [19], sol–gel-derived ceramics [20], etc. to get different modified electrodes for protein electrochemistry. Recently room temperature ionic liquids (RTILs) have attracted great attentions in the field of chemistry due to their excellent properties such as negligible vapor pressure, high chemical and thermal stability, good conductivity and dissolving capability. The reviews about the application of RTILs in the fields of analytical chemistry or electrochemistry had been reported [21,22]. Due to the advantages such as higher ionic conductivity and wider electrochemical windows, RTILs can be used as the modifier or the supporting electrolyte in the field of electroanalysis. Wei and Ivaska reviewed the recent progress of RTILs in the electrochemical sensors [23]. Compared with other modified electrodes, ionic liquid modified carbon paste electrode (CILE) shows some advantages such as higher sensitivity, wider electrochemical windows and good anti-fouling ability [24,25]. So CILE had been used as the basal electrode for the investigation on the redox proteins electrochemistry or applied to the detection of some electroactive substances [26–30]. Wang et al. also applied the RTILs as the electrolyte for the investigation of the direct electrochemistry of heme proteins [31,32]. In this paper, a CILE was first fabricated by using a hydrophilic ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 ) as modifier and further modified with multiwalled carbon nanotubes (MWCNTs). Hemoglobin was immobilized on the surface of MWCNTs/CILE and subsequently fixed by a layer of polyvinyl alcohol (PVA) film. PVA is a polymer composed of a carbon chain backbone attached with hydroxyl groups, which can provide a biocompatible microenvironment for proteins or enzyme immobilization and improve the stability of the modified electrode. The modified electrode was characterized by different methods such as scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The direct electron transfer of Hb with the modified electrode was achieved and the results indicated that the MWCNTs/CILE could effectively facilitate the direct electrochemistry of Hb. The electrocatalysis of the resulting electrode to the reduction of hydrogen peroxide (H2 O2 ) and trichloroacetic acid (TCA) were further studied. 2. Experimental 2.1. Reagents Ionic liquid 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 , Hangzhou Kemer Chemical Limited Company), bovine hemoglobin (Hb, MW. 64500, Tianjin Chuanye Biochemical Limited Company), multi-walled carbon nanotubes (MWCNTs, purity >95%, main range of diameter <10 nm, length of 5–15 m, Shenzhen Nanoport Company, China), polyvinyl alcohol (PVA, average degree of polymerization 7500, MW. 14,000, Tianjin Bode Chemical Limited Company), graphite powder (average particle size 30 m, Shanghai Colloid Chemical Company), trichloroacetic acid (TCA, Tianjin Kemiou Chemical Limited Company) were used as received. 0.1 mol/L 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 all the experiments. 2.2. Apparatus A CHI 750B electrochemical workstation (Shanghai CH Instrument, China) was used for all the electrochemical measurements
including cyclic voltammetry and electrochemical impedance spectroscopy. A conventional three-electrode system was used with a Hb modified electrode as working electrode, a platinum wire as auxiliary electrode and a saturated calomel electrode (SCE) as reference electrode. UV–vis absorption spectra and FT-IR spectra were recorded on Cary 50 probe spectrophotometer (Varian Company, Australia) and Tensor 27 FT-IR spectrophotometer (Bruker Company, Germany), respectively. Scanning electron microscopy was performed on a JSM-6700F scanning electron microscope (Japan Electron Company, Japan). 2.3. Preparation of PVA/Hb/MWCNTs/CILE CILE was fabricated with the following procedure: 0.15 mL of EMIMBF4 , 0.85 mL of liquid paraffin and 3.2 g of graphite powder were hand-mixed in an agate mortar and ground carefully. A portion of resulting homogeneous paste was packed firmly into a glass tube cavity (˚ = 4 mm). The electric contact was established through a copper wire to the end of the paste in the inner hole of the tube and the surface of CILE was polished by smoothing on a weighing paper. The traditional carbon paste electrode was prepared by mixing graphite powder with liquid paraffin at a ratio of 70/30 (w/w) according to the general procedure. The MWCNTs modified CILE (MWCNTs/CILE) was prepared by simply applying 3.0 L of 1.0 g/L MWCNTs suspension solution (dispersed in dimethyl formamide, DMF) on the surface of CILE and left it to dry at the room temperature for 2 h. Then 10.0 L of 20.0 mg/mL Hb solution was dropped on the surface of MWCNTs/CILE and stayed silent to allow the water evaporated gradually. At last 10.0 L of 2.0 mg/mL PVA solution was cast on the surface of electrode to form a stable film. During these procedures a small bottle was fit over the electrode so that the solvent could evaporate slowly to get a uniform film. The resulted modified electrode was denoted as PVA/Hb/MWCNTs/CILE and stored at 4 ◦ C when not in use. Other modified electrodes including PVA/hemin/MWCNTs/CILE, PVA/Hb/MWCNTs/CPE, PVA/MWCNTs/CILE, etc. were prepared by the similar procedures and used in the experiment for comparison. 2.4. Procedure The electrochemical measurements were carried out in a 10-mL electrochemical cell containing 0.1 mol/L PBS, which was purged with highly purified nitrogen for 30 min prior to a series of experiments and maintained in a nitrogen atmosphere during the experiments. UV–vis spectroscopic experiments were performed with a mixture solution of certain concentration of Hb, MWCNTs and PVA with different pH PBS. The PVA/Hb/MWCNTs film assembled on a glass slide was used for FT-IR measurements. 3. Results and discussion 3.1. Spectroscopic characterizations In UV–vis absorption spectrum the Soret absorption band from the four iron heme groups of heme proteins may provide the information on the conformational integrity of the proteins and the possible denaturation or the conformational change of heme region [33]. As shown in Fig. 1, Hb had a Soret band at 406.0 nm in water (curve a). After mixing Hb, PVA and MWCNTs with different external pH buffer solutions as 5, 7 and 10, respectively, the absorption value also appeared at 406.0 nm (curve b–d). Obviously, these absorption peaks were attributed to the Soret band of Hb and the results suggested that Hb in the composite film retained its native structure in a wide range of buffer pH. While in a pH 2.0 buffer solution, the
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Fig. 1. UV–vis absorption spectra of Hb in water (a) and PVA/Hb/MWCNTs with pH (b) 5.0 (c) 7.0, (d) 10.0, and (e) 2.0 PBS, respectively.
Soret band became broader and smaller with the maximum absorption band at 368.0 nm (curve e), indicating that Hb molecules were denatured to a certain extent in the acidic solution. FT-IR spectroscopy is also a sensitive method to probe into the secondary structure of proteins. The profiles of the amide I and amide II infrared absorbance bands of Hb provide detailed information on the secondary structure of the polypeptide chain. The amide I band at 1700–1600 cm−1 is attributed to the C O stretching vibra-
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Fig. 2. FT-IR spectra of (a) Hb and (b) PVA/Hb/MWCNTs film.
tion of the peptide linkage in the backbone of protein. The amide II band at 1600–1500 cm−1 is caused by the combination of N H inplane bending and C N stretching vibration of the peptide groups [34]. If Hb molecule is denatured, the intensity and shape of the amide I and amide II bands will diminish or even disappear [35,36]. As shown in Fig. 2, the FT-IR spectra of amide I and II bands of native Hb were located at 1655.34 and 1535.80 cm−1 , respectively (Fig. 2a). After mixing Hb, PVA and MWCNTs together, the spectra of amide I and amide II bands appeared at 1653.55 and 1538.66 cm−1 (Fig. 2b).
Fig. 3. SEM images of (a) CILE, (b) MWCNTs/CILE, (c) Hb/MWCNTs/CILE and (d) PVA/Hb/MWCNTs/CILE.
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The similarities of the two spectra suggested that Hb retained the essential features of its native structure after immobilized in the PVA and MWCNTs composite films. 3.2. Surface morphologies of modified electrode Scanning electron microscopy was used to characterize the top views of the different modified electrodes and the results were shown in Fig. 3. On CILE (Fig. 3a) an uniform surface appeared without separated carbon layer, which was due to the embedment of ionic liquids EMIMBF4 between the layer of carbon and disperse the carbon powder homogeneously. While on the MWCNTs/CILE (Fig. 3b), there were plenty of tubes dispersed on the surface, which indicated that MWCNTs were successfully immobilized on the surface of CILE. After MWCNTs/CILE was further coated with Hb molecules, the aggregation of the immobilized Hb molecules was distributed regularly and showed a network-like structure (Fig. 3c). On the PVA/Hb/MWCNTs/CILE (Fig. 3d) an unshaped membrane appeared, which was due to the presence of PVA film on the electrode surface. 3.3. Characteristics of the modified electrodes Electrochemical impedance spectroscopy can provide information on the impedance changes of the electrode surface during the modification process. By using [Fe(CN)6 ]3−/4− redox couples as the electrochemical probe, the Nyquist plots of different modified electrodes were shown in Fig. 4. On the bare CILE, the electron transfer resistance (Ret), which was derived from the semicircle domains of impedance spectra, can be estimated to be 667.4 (curve a). After PVA was coated on the surface of CILE, the Ret value was increased to 1102.3 (curve b), which indicated a film of polymer PVA formed on the surface could hinder the electron transfer. While on MWCNTs/CILE, the Ret value was decreased dramatically and nearly to zero (curve c), indicating that MWCNTs present on the surface of CILE could enhance the conductivity. Then the Ret value increased to 83.7 after the PVA was coated on the MWCNTs/CILE (curve d). Subsequently, when Hb was immobilized on the PVA/MWCNTs/CILE the Ret value increased again (curve e), which indicated that Hb molecules were successfully immobilized in the PVA/MWCNTs films. Cyclic voltammetric results of ferricyanide with different modified electrodes are also a valuable and convenient tool to
Fig. 4. EIS for (a) bare CILE, (b) PVA/CILE, (c) MWCNTs/CILE, (d) PVA/MWCNTs/CILE and (e) PVA/Hb/MWCNTs/CILE in a mixture solution of 10.0 mmol/L [Fe(CN)6 ]3−/4− and 0.1 mol/L KCl with the frequencies swept from 105 to 0.1 Hz under a open circuit potential conditions.
Fig. 5. Cyclic voltammograms of (a) bare CILE, (b) PVA/CILE, (c) MWCNTs/CILE, (d) PVA/MWCNTs/CILE and (e) PVA/Hb/MWCNTs/CILE in a mixture solution of 1.0 mmol/L [Fe(CN)6 ]3− and 0.1 mol/L KCl with the scan rate as 100 mV s−1 .
monitor the modification process on the modified electrodes. Cyclic voltammograms of different modified electrodes with 1.0 mmol/L [Fe(CN)6 ]3− solution were recorded with the results shown in Fig. 5. A pair of well-defined redox peaks was observed at the bare CILE (curve a). When PVA was coated on the bare CILE, the redox peak currents decreased drastically (curve b), implying the presence of PVA film on the electrode surface acted as an inert layer and blocked the diffusion of ferricyanide toward the electrode. However, on the MWCNTs/CILE, the electrochemical response was bigger than that of bare CILE (curve c), which indicated that the presence of MWCNTs could effectively improve the conductivity of the electrode. When PVA was coated on the MWCNTs/CILE, the redox peak current decreased correspondingly (curve d). After Hb was further immobilized in the PVA/MWCNTs/CILE, the redox peak current was further decreased (curve e), which indicated that Hb molecules in the film further blocked the diffusion of ferricyanide. The data were in consistent with the results obtained from EIS experiments. On the basis of the EIS and cyclic voltammetric results, we can conclude that Hb was successfully immobilized in the PVA/MWCNTs composite films on the surface of CILE. 3.4. Direct electrochemistry of Hb Fig. 6 shows the typical cyclic voltammograms of different electrodes in pH 7.0 PBS at the scan rate of 200 mV s−1 . No electrochemical responses were observed at bare CILE (curve a), PVA/CILE (curve b) and MWCNTs/CILE (curve c), which indicated no electroactive substances existed on the electrode surface. While on the PVA/Hb/CILE a pair of small and unsymmetric redox peaks appeared (curve d), which indicated that the direct electron transfer of Hb with CILE had taken place with slow electron transfer rate, and the quasi-reversible electrochemical responses appeared. Since CILE had the advantages of high conductivity, high sensitivity, good anti-fouling ability and a layer of IL was presented on the surface of CILE [24,25], it can provide a suitable interface to slightly promote the electron transfer reaction of protein. While on the PVA/Hb/MWCNTs/CILE, the electrochemical response of Hb was greatly enhanced and a pair of well-defined quasi-reversible cyclic voltammetric peaks was observed (curve f). For comparison the cyclic voltammogram of PVA/Hb/MWCNTs/CPE was also recorded (curve e). Only a small reduction peak appeared without the oxidation peak, indicating that the direct electron transfer process was not realized on the MWCNTs modified CPE. Although MWCNTs have many specific advantages such as large surface area,
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trode for the reduction or oxidation of electroactive species in the thin films. According to the Q– * relationship equation: Q = nFA * [41], the surface concentration ( *) of electroactive Hb in the film was calculated with the average value as 3.5 × 10−9 mol cm−2 . The results were larger than the theoretical monolayer value (1.89 × 10−11 mol cm−2 ) of protein [31], indicating that several layers of protein could exchange electrons with the electrode, which may be due to the presence of MWCNTs on the electrode surface resulted in a three-dimensional structure for Hb immobilization. The total amount of Hb in the modified film was calculated as 2.47 × 10−8 mol cm−2 based on the volume and the concentration of Hb solution deposited on the electrode surface. So the fraction of electroactive Hb was calculated as 14.2%. With the increase of the scan rate the peak-to-peak separation (Ep) were also increased gradually, so the electrochemical parameters could be calculated using the method developed by Laviron [42,43], which was valid for flat film modified electrode with surface-controlled electrochemical process at the value of Ep < 200 mV: Fig. 6. Cyclic voltammograms of (a) bare CILE, (b) PVA/CILE, (c) MWCNTs/CILE, (d) PVA/Hb/CILE, (e) PVA/Hb/MWCNTs/CPE and (f) PVA/Hb/MWCNTs/CILE in pH 7.0 PBS at the scan rate of 200 mV s−1 . Inset: cyclic voltammogram of PVA/hemin/MWCNTs/CILE in pH 7.0 PBS at the scan rate of 200 mV s−1 .
Epc = E 0 −
high biocompatible interface and good electric conductivity, the presence of CILE also played important roles in realizing the direct electron transfer process. So the synergistic effect of CILE and MWCNTs played important roles in achieving the redox reaction of Hb in the composite film. As indicated in references [37–39], a layer of IL existed on the surface of CILE and MWCNTs could interact with the imidazolium group on the electrode surface through – or/and -cationic, hydrophobic and electrostatic interaction. Then a stable MWCNT–IL composite film was formed on the surface of electrode for the further Hb immobilization. From the cyclic voltammogram the redox peak potentials were got with the cathodic peak potential (Epc) as −0.436 V and the anodic peak potential (Epa) as −0.303 V (vs. SCE). The formal potential (E0 ), 0 which is calculated from the equation as E = (Epa + Epc)/2, was estimated as −0.370 V (vs. SCE) and it was the characteristic of heme FeIII /FeII redox couples [40]. The peak-to-peak separation (Ep) at 200 mV s−1 was got as 133 mV and the ratio of redox peak current (Ipa/Ipc) was approximately to be 1. The results indicated a quasi-reversible electrochemical behavior. Also the cyclic voltammogram of PVA/hemin/MWCNTs/CILE was further investigated (insert of Fig. 6). A pair of small and unsymmetric redox peaks with Epc as −0.547 V and Epa as −0.348 V (vs. SCE) appeared. The formal potential (E0 ) was estimated as −0.448 V (vs. SCE). The results were different with that of PVA/Hb/MWCNTs/CILE, indicating that the Hb molecules in the composite film was not denatured or degenerated. Cyclic voltammograms of PVA/Hb/MWCNTs/CILE with different scan rates from 50 to 800 mV s−1 in pH 7.0 PBS were further recorded. A pair of quasi-reversible redox peaks appeared with the almost equal high of the redox peak current, which indicated that all the electroactive heme FeIII of the protein in the film was reduced to heme FeII in the forward cathodic scan with the complete conversion of heme FeII back to its FeIII form in the reversed anodic scan. Both the redox peak currents increased with the scan rate and two linear regression equations were got as Ipc (A) = 0.0054 (V s−1 ) + 0.558 with a correlation coefficient () of 0.999 and Ipa (A) = −0.0058 (V s−1 ) −0.326 ( = 0.995), which indicated a typical characteristic of diffusionless surface-controlled thin-layer electrochemical behavior. For a thin-layer electrochemistry, the integration of cyclic voltammetric peaks of protein film electrode in the buffer gives the total amount of charge value (Q) passed through the elec-
Epa = E 0 +
RT ln ˛nF
(1)
RT ln (1 − ˛)nF
(2)
log ks = ˛ log (1 − ˛) + (1 − ˛) log ˛ − log nFEp − ˛(1 − ˛) 2.3RT
2.3RT nF (3)
where ˛ is the electron transfer coefficient, n is the electron transfer number, ks is the apparent heterogeneous electron transfer rate constant, R is the gas constant, T is the absolute temperature, and Ep is the peak-to-peak separation. The relationship of Epc and Epa with ln were constructed with the linear regression equations as Epa (V) = 0.0216 ln − 0.268 ( = 0.996) and Epc (V) = −0.041 ln − 0.502 ( = 0.994). According to Eqs. (1) and (2) the electron transfer coefficient (˛) was calculated as 0.49. Based on Eq. (3) the apparent heterogeneous electron transfer rate constant (ks ) was further calculated from the relationship of Ep with ln and the result was got as 1.054 s−1 , which was higher than some previous reports with casting method for protein immobilization [44–46], indicating that the PVA/MWCNTs/CILE provided a suitable microenvironment for the increase of electron transfer rate of Hb. But the value was smaller than most of the Hb biosensor fabricating by layer-by-layer method, which may be due to the different preparation methods provide different microenviroment for Hb molecules to transfer electron [47].
3.5. Effect of buffer pH The direct electrochemistry of the Hb immobilized in PVA/MWCNTs film showed a strong dependence on pH value of external solutions with the cyclic voltammograms shown in Fig. 7. An increase of buffer pH led to the negative shift of both the reduction and oxidation peak potentials. The formal potential (E0 ) had a linear relationship with pH value from 4.0 to 10.0 with the linear regression equation as E0 (V) = −0.0422 pH −0.0305 ( = 0.998). The value of slope was −42.2 mV/pH, which was reasonably smaller to the theoretical value of −56.0 mV/pH at 20 ◦ C for the reversible one-electron transfer coupled with single-proton transportation. But the electrode process can also be represented as Hb FeIII + H+ + e Hb FeII , where the charges of Hb molecules are omitted [48].
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W. Sun et al. / Electrochimica Acta 54 (2009) 4141–4148
Fig. 7. Influence of pH on cyclic voltammograms of PVA/Hb/MWCNTs/CILE with 4.0, 6.0, 8.0, and 10.0 of the pH values, respectively, at the scan rate of 200 mV s−1 .
3.6. Electrocatalytic reactivity The PVA/Hb/MWCNTs/CILE showed good electrocatalytic ability to different substrates such as H2 O2 and trichloroacetic acid. As shown in Fig. 8, with the addition of H2 O2 into the pH 7.0 PBS an obvious increase of the cathodic peak current at −0.436 V (vs. SCE) appeared with the decrease of the anodic peak current (curves a–f). However no similar phenomena could be observed at other modified electrodes without the Hb under the same conditions. The more the H2 O2 added, the larger the cathodic peak current got, which indicated the Hb incorporated in the composite film could act as an effective catalyst to the reduction of H2 O2 . A linear relationship between the catalytic peak current and H2 O2 concentration was obtained (inset of Fig. 8) in the range from 1.2 to 30.0 mol/L with the linear regression equation as Ip (A) = 0.286 C (mol/L) + 0.256 ( = 0.997) and the detection limit as 1.0 mol/L (3). The relative standard deviation (R.S.D.) for the six successive determinations of 4.0 mol/L H2 O2 was calculated as 3.7%. When the concentration of H2 O2 was more than 50.0 mol/L, a plateau of catalytic peak current appeared, which suggested a typical electrocatalytic process with Michaelis–Menten kinetic model. The apparent Michaelis–Menten app constant (KM ) was further calculated from the electrochemical
Fig. 9. Cyclic voltammograms of PVA/Hb/MWCNTs/CILE in 0.1 mol/L pH 7.0 PBS containing 0, 0.8, 3.6, 4.4, 6.0, 8.0, and 10.0 mmol/L TCA (curves a–h), respectively, with the scan rate of 100 mV s−1 .
version of the Lineweaver–Burk equation [49], which could provide an indicative of the enzyme–substrate kinetics. app
K 1 1 = + M Iss Imax Imax c
(4)
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 the saturated substrate condition. By an analysis the slope and the intercept of the plot of the reciprocals of the reduction peak current versus H2 O2 concentration, the app KM value was calculated as 0.928 mmol/L, which was smaller than app some reported H2 O2 biosensors [50,51]. The smaller KM value indicated that the higher activity of immobilized Hb in the composite film. Electrocatalytic activity of PVA/Hb/MWCNTs/CILE towards TCA was also investigated with the results shown in Fig. 9. When TCA was added into a pH 7.0 PBS, an increase in the Hb FeIII reduction peak current was observed with the decrease of Hb FeII oxidation peak. This may be due to the reaction of Hb FeII with TCA to produce Hb FeIII again, which then reduced electrochemically at electrode to form a catalytic cycle. The catalytic reduction peak current increased with the TCA concentration in the range from 0.5 to 12.0 mmol/L and the linear regression equation was got as Ip (mA) = 0.0322 C (mmol/L) + 0.0015 ( = 0.998) with the detection app limit as 0.1 mmol/L (3). According to Eq. (4) the KM value of the enzyme–substrate kinetic reaction was calculated as 5.13 mmol/L, which was lower than that of the previous report [48]. 3.7. Stability and repeatability of the modified electrode
Fig. 8. Cyclic voltammograms of PVA/Hb/MWCNTs/CILE in pH 7.0 PBS containing 0, 1.0, 3.0, 4.0, 5.0, and 20.0 mol/L H2 O2 (from a–f), respectively, at the scan rate of 100 mV s−1 . Inset: the relationship between the peak current and the concentration of H2 O2 .
The stability of the CILE was first investigated. Since EMIMBF4 was a hydrophilic ionic liquid which is totally water miscible, it was used as modifier in the carbon paste electrode. The ratio of liquid paraffin and EMIMBF4 was optimized, which showed great influence on the stability of the CILE. The peak current remained stable in the ratio range from 9/1 to 4/1. When the ratio was more than 7/3, the peak current tended to be unstable due to the partly dissolution of EMIMBF4 into the solution. So the final volume ratio of liquid paraffin and EMIMBF4 was selected as 85/15, which gave stable response for 100 continuous scanning in pH 7.0 PBS. The storage of the CILE also showed good stability in 1 month without the changes of the background currents in the same buffer solution.
W. Sun et al. / Electrochimica Acta 54 (2009) 4141–4148
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Table 1 Comparison of different redox protein film electrodes for H2 O2 detection. Modified electrode Hb/IL/CILE Hb–Nafion–Co3 O4 –GCE SA/Hb/CILE HRP-PEG/PGE HRP-Chi-BMIMBF4 /GCE Hb-CILE PVA/Hb/MWCNTs/CILE
E0 (V)
* (mol cm−2 )
−0.22 −0.350 −0.344 −0.379 −0.34 −0.334 −0.370
8.4 × 10−11
3.1 × 10−9 3.2 × 10−10 – 1.03 × 10−10 1.38 × 10−9 3.5 × 10−9
Linear range (M) 100–5000 1–390.0 1.0–100.0 2.0–100.0 0.75–135.0 8.0–280.0 1.2–30.0
app
Detection limit (M)
KM (mM)
Reference
40 0.1 1.0 67 0.25 1.0 0.5
7.5 0.136 – 1.38 – 1.103 0.928
[30] [52] [53] [54] [55] [56] This paper
GCE: glassy carbon electrode; SA: sodium alginate; PEG: polyethylene glycol; PGE: pyrolytic graphite electrode; Chi: chitosan; BMIMBF4 : 1-butyl-3-methylimidazolium tetrafluoroborate; MWCNTs: multiwall carbon nanotubes.
Then the stability of PVA/Hb/MWCNTs/CILE was further investigated with two methods. Firstly the modified electrode was evaluated by examining the cyclic voltammetric peak currents after continuous scanning for 50 cycles. There was nearly no decrease of the voltammetric response, indicating that PVA/Hb/MWCNTs/CILE was stable in buffer solution. The storage stability of the Hb modified electrode was investigated by keeping the electrode at 4 ◦ C when the electrode was not in use. 95.3% of the initial current response was retained after 2 weeks storage. After 1 month of testing, the peak current response decreased about 10%. The relatively good stability of the Hb electrode can be attributed to the biocompatibility between the PVA/MWCNTs composite film and Hb. The composite film can prevent the leakage of the proteins and retain electrocatalytic activity of the proteins. Six Hb modified electrodes were made by the same procedure independently and the relative standard deviation of 4.3% was calculated for the determination of 4.0 × 10−6 mol/L H2 O2 , which indicated the modified electrode had good repeatability. 4. Conclusion By using a hydrophilic ionic liquid EMIMBF4 , MWCNTs and PVA as the composite materials, Hb was successfully immobilized on a multi-walled carbon nanotubes modified carbon ionic liquid electrode. The resulting PVA/Hb/MWCNTs/CILE showed good electrochemical behaviors and a pair of well-defined quasi-reversible redox couple of Hb FeIII /FeII was got with the formal potential as −0.370 V (vs. SCE) in pH 7.0 PBS. The results indicated that the synergistic effect of MWCNTs and CILE played important roles in facilitating the direct electron transfer of Hb. The electrochemical parameters of Hb in the film were carefully calculated and the Hb modified electrode showed good electrocatalytic ability to the reduction of H2 O2 and TCA. The comparison of the proposed electrode with other redox protein modified electrodes fabricated with Nafion or other ionic liquids was listed in Table 1. Compared with the another commonly used hydrophobic ionic liquid 1-butyl-3methylimidazolium hexafluorophate (BMIMPF6 ) used for protein app modified electrode, the lower detection limit and small KM value appeared, which may be due to the hydrophilic solvent have strong effect on the activity of the protein. Also EMIMBF4 showed higher conductivity and smaller viscosity than BMIMPF6 . So the electron transfer process was much faster with EMIMBF4 as the modifier. As for the different film forming materials including Nafion and chitosan, it was difficult to evaluate the performance because of different basal electrode or nanomaterials were used. But the high hydrophilic ability of PVA could provide a suitable microenvironment for Hb to keep the bioactivity. So the PVA/Hb/MWCNTs/CILE may have the future application in the third-generation biosensors. Acknowledgements We are grateful to the financial support of the National Science Foundation of China (Nos. 20635020 and 20405008) and the
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