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Direct electrochemistry of myoglobin based on DNA accumulation on carbon ionic liquid electrode
Electrochemistry Communications 11 (2009) 1527–1529
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Direct electrochemistry of myoglobin based on DNA accumulation on carbon ionic liquid electrode Ruifang Gao, Jianbin Zheng * Institute of Analytical Science, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, PR China
a r t i c l e
i n f o
Article history: Received 5 May 2009 Received in revised form 21 May 2009 Accepted 22 May 2009 Available online 29 May 2009 Keywords: Electroanalysis Direct electrochemistry Electrostatic interaction Ionic liquid Myoglobin Deoxyribonucleic acid
a b s t r a c t Poly-anionic deoxyribonucleic acid (DNA) was accumulated on the positively charged surface of carbon ionic liquid electrode (CILE) with N-butylpyridinium hexafluorophosphate (BPPF6) as binder, and then myoglobin (Mb) was immobilized onto the DNA film by electrostatic interaction to form Mb/DNA/CILE electrode. The direct electrochemistry of Mb was then investigated in detail. A pair of well-defined, quasi-reversible cyclic voltammetric peaks of Mb was obtained with the formal potentials (E00 ) at 0.304 V (vs. SCE) in phosphate buffer solution (PBS, pH 7.0). The Mb/DNA/CILE electrode showed excellent electrocatalytic activity to H2O2 and trichloroacetic acid (TCA) in the range of 1.0–160 lmol/L and 0.5–40.0 mmol/L, respectively. The apparent Michaelis–Menten constants (KM) toward H2O2 and TCA were calculated as 0.42 and 0.82 mmol/L. So, the DNA/CILE had potential to study other proteins. Ó 2009 Published by Elsevier B.V.
1. Introduction Different materials for immobilization enzymes on the surface of working electrode have been employed including sol-gel, polymers, nanoparticles [1–5]. Among them, deoxyribonucleic acid (DNA) as an important biological macromolecule has electrical conducting electrochemical properties and unique three dimensional structures. Immobilized DNA on the surface of a substrate electrode not only provides a biocompatible microenvironment for protein, but also dramatically increases the coverage of protein on the electrode surface [6]. So, more and more attention was paid on the application of DNA to biosensors. Fan et al. [7] adopted cast method to immobilize hemoglobin and DNA on pyrolytic graphite electrode and a nitric oxide biosensor was fabricated. Wang et al. [8] successfully immobilized multi-walled carbon nanotube on the surface of platinum electrode by mixing with DNA, and then cytochrome c was adsorbed onto the modified electrode to investigate its direct electrochemistry. Recently, room temperature ionic liquids (RTILs) have been employed as a new media in electrochemistry and electroanalysis. As green solvent with many specific physicochemical properties such as high ionic conductivity, wide electrochemical range, negligible vapor pressure and thermal stability, RTILs have been a promising choice not only for supporting electrolyte but also for the modified materials incorporating with conventional biocomposite matrixes * Corresponding author. Tel.: +86 29 88302077; fax: +86 29 88303448. E-mail address: [email protected] (J. Zheng). 1388-2481/$ - see front matter Ó 2009 Published by Elsevier B.V. doi:10.1016/j.elecom.2009.05.046
such as carbon materials, cellulose, chitosan, silica sol-gel [9–11] to investigate direct electrochemistry of redox proteins. RTILs as a binder were also widely used in biosensors. Maleki et al. [12] fabricated a high-performance carbon composite electrode (CILE) by using graphite powder mixed with n-octylpyridinium hexafluorophosphate as binder, the resulted CILE showed attractive electrochemical behaviors. Sun et al. [13] also combined N-butylpyridinium hexafluorophosphate (BPPF6) with graphite powder to make a new kind of CILE. Then, they successfully fabricated several protein biosensors layer-by-layer method with this CILE [14,15]. The aim of this work was to preparation a controllable thin DNA film on the surface of CILE (DNA/CILE) by electrochemistry method. Then, Mb was immobilized on DNA/CILE to investigate its behavior of direct electrochemistry on the DNA film. 2. Experimental 2.1. Reagents Bovine myoglobin (Mb, MW. 64500) and Salmon sperm dsDNA were from Sigma. N-butylpyridinium hexafluorophosphate (BPPF6, 97%) was purchased from Hangzhou Kemer Chemical Limited Company. Graphite powder (SP) was got from Sinopharm Chemical Reagent Co. Ltd. H2O2 (w/w, 30%) was got from Tianjin Tianda Chemistry Reagent Plant. TCA was from Shanghai Shanpu chemical plant Co. Ltd. 0.1 mol/L phosphate buffer solution (PBS) was used as the supporting electrolyte. Other reagents were analytical
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reagent grade and doubly distilled water was used in all the experiments. 2.2. Apparatus All the electrochemical experiments were carried out on a CHI660A electrochemical workstation (Shanghai CH Instrument Co. Ltd., China) using a three electrode system. The working electrode was made by the following procedure. A saturated calomel electrode (SCE) and a platinum electrode were served as reference and counter electrode, respectively. All the tested solutions were purged with highly purified nitrogen for 30 min before the experiments and a nitrogen environment was kept over the solution during the electrochemical measurements. 2.3. Preparation of Mb modified electrode CILE was prepared under the optimum condition: 1.2 g of graphite powder and 1.0 g of BPPF6 were mixed thoroughly in a mortar and heated at 80 °C for about 2 h to form a homogenous carbon paste. Then, a glass tube (U = 3 mm) and a copper wire were applied to fabricate the CILE. After carefully polishing the surface of CILE on a piece of weighting paper, the following procedure was performed to fabricate an Mb/DNA/CILE electrode. Firstly, a bare CILE was immersed in pH 7.0 PBS solution containing 2.0 mg/mL DNA at the potential of +1.5 V for 360 s. Then, the obtained DNA/CILE electrode was incubated in 10 mg/mL Mb solution at pH 5.0 for 10 h to fabricate the Mb/DNA/CILE electrode. DNA/ CILE and Mb/CILE were prepared in the same way.
3.2. Direct electrochemistry of Mb The CVs of the DNA/CILE, Mb/CILE and Mb/DNA/CILE modified electrodes were measured in 0.1 M PBS (pH 7.0) at 100 mV/s (Fig. 2). At DNA/CILE (curve a), no peak was found and a high background current was observed, which was due to the high capacitance of RTIL and it was the typical characteristic of CILE [12,16,17]. Curve b was the voltammogram of Mb/CILE, a very small redox peaks was appeared. After DNA accumulating on CILE as the film to adsorb Mb, a remarkable enhanced electrochemical response was observed (curve c). A pair of well-defined, quasireversible redox peaks of Mb were located at 0.336 and 0.271 V with the DEp of 65 mV and formal potential (E00 = (Epa + Epc)/2) of 0.304 V (vs. SCE), respectively. The electron transfer reactivity of Mb was significantly improved on the DNA film. It may be attribute to that the immobilization of DNA on electrode surface can form a conductive thin layer of nano-structures to further enhance the electrode surface area for immobilizing more Mb [6]. Cyclic voltammetry was applied to investigate the behaviors Mb/DNA/CILE in pH 7.0 PBS at different scan rate. Both the cathodic and anodic peak currents of Mb increased linearly with scan rates from 0.05 to 0.6 V/s, indicating that a typical surface controlled electrochemical process was achieved on Mb/DNA/CILE [14]. The anodic and cathodic peak potentials were linearly dependent on the logarithm of the scan rates with slopes of 2.3RT/anF and 2.3RT/(1a)nF [14]. So, the charge-transfer coefficient (a) was calculated as 0.57 and the number of electron transferred was 0.94 1, respectively. According to the equation model of Laviron [18], the electron transfer rate constant (ks) was further estimated to be 1.02 s1.
3. Results and discussion 3.3. Electrocatalytic behaviors to H2O2 and TCA 3.1. Characterization of DNA film The electrochemical behaviors of different modified electrodes 3=4 solution containing 0.1 M KCl were charactered by FeðCNÞ6 and the cyclic voltammograms (CVs) were shown in Fig. 1. At bare CILE (curve a), a reversible electrochemical response for FeðCNÞ63=4 was observed with a peak-to-peak separation (DEp) of 66 mV at 100 mV/s. After accumulation DNA on the bare CILE, both the cathodic and anodic peak currents decreased obviously and the DEp increased to 70 mV (curve b). It indicated that the DNA immobilized on the electrode had slowed down the electron transfer of the redox probe due to the electrostatic repulsion between the poly-anionic DNA and the redox couple ions carrying negative charge [6]. When Mb was immobilized on DNA/CILE, a further decrease of the peak currents of FeðCNÞ63=4 was obtained with DEp of 77 mV (curve c), which was due to the non-conductive properties of Mb that acted as an inert electron layer and hindered the electron transfer.
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The electrocatalytic reduction of H2O2 and TCA was studied by cyclic voltammetry. The catalytic reduction of H2O2 on Mb/DNA/ CILE electrode was first examined. As shown in Fig. 3A, after adding H2O2 into the deoxygenated pH 7.0 PBS, a new reduction peak appeared at about 0.368 V (vs. SCE). While, a reduction peak potential more than 0.7 V was obtained on the DNA/CILE with the addition of H2O2 (curve b). Thus, the reduction potential of H2O2 decreased about 0.4 V, indicating the presence of Mb in the DNA film decreased the reaction activation energy observably. With increasing the concentration of H2O2, the reduction peak current of Mb increased dramatically, while the anodic peak current decreased to almost zero (curve d–n), which demonstrated the typical electrocatalytic reduction process of H2O2 [19]. The reduction peak current had a linear relationship with the concentration of H2O2 in the range from 1.0 to 160 lmol/L. The linear regression equation was Ipc (lA) = 0.711 C (lmol/L) + 11.49 (n = 34, r = 0.9975) with a detection of 0.2 lmol/L (S/N = 3).
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Fig. 1. CVs obtained at (a) bare CILE, (b) DNA/CILE and (c) Mb/DNA/CILE in 1 mmol/ solution containing 0.1 mol/L KCl. Scan rate: 100 mV/s. L FeðCNÞ3=4 6
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Fig. 2. CVs of (a) DNA/CILE, (b) Mb/CILE, (c) Mb/DNA/CILE in pH 7.0 PBS at scan rate of 100 mV/s.
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Fig. 3. (A) CVs of DNA/CILE in the presence of (a) 0, (b) 100.0 lmol/L H2O2, (c–n) Mb/DNA/CILE in the presence of 0, 8.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 120.0 lmol/L H2O2, respectively. Inset: the linear part of the calibration curve. (B) CVs of DNA/CILE in the presence of (a) 0, (b) 2.0 mmol/L TCA, (c–l) Mb/DNA/CILE in the presence of 0, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 mmol/L TCA, respectively. Inset: relationship of the catalytic peak current and the concentration of TCA. Scan rate: 100 mV/s.
When the concentration of H2O2 was more than 1.6 104 mol/L, a response plateau was observed, showing a typical Michaelis–Menten kinetic mechanism. The enzyme-substrate kinetics could be indicated by the apparent Michaelis–Menten constant (KM), which can be obtained from the electrochemical version of Lineweaver–Burk equation [20]. So, the KM value was calculated as 0.42 mmol/L, which was lower than some previous reports [21,22], suggesting that Mb retained higher activity in the DNA film on the surface of CILE. Electrochemical catalytic reduction of TCA on the Mb/DNA/CILE was also investigated (Fig. 3B). After the addition of TCA into a pH 7.0 PBS, the reduction peak increased with the decrease of the oxidation peak significantly. The more the TCA was added, the greater the reduction peak current was increased (curve d–l). The direct reduction of TCA at DNA/CILE was not found in the potential range of 0.1 to 1.0 V. This indicated that the presence of Mb could reduce the over potential of TCA. The reduction peak current is linearly proportional to the concentration of TCA in the range of 0.5– 40.0 mmol/L (Fig. 3B inset) with the linear regression as Ipc(lA) = 1.06 C (104 mol/L) + 9.46 (n = 18, r = 0.9973) and the detection limit was 8.3 105 mol/L. In addition, according to the electrochemical version of Lineweaver–Burk equation [20], the KM was calculated as 0.82 mmol/L, which was lower than some previous reports [23,24], showing a high affinity to TCA. 3.4. Reproducibility and stability of the Mb/DNA/CILE The Mb modified electrode was used for 11 parallel determinations of 50.0 lmol/L H2O2 solution and the relative standard deviation (RSD) was got as 4.2%. The stability of the Mb modified electrode was evaluated by 80 continuous cyclic scans. No obvious decrease of the voltammetric response was found. After storing two weeks at 4 °C refrigerator, the modified electrode nearly remained its initial current response. After one-month storage, the modified electrode retained about 91.6% of the initial current response. The results indicated that DNA film was efficient for Mb immobilization and the bioactivity of Mb was remained. 4. Conclusions Poly-anionic DNA was accumulated on the positively charged surface of CILE electrode to form a conductive thin layer film of DNA, and then Mb was immobilized onto the film by electrostatic interaction to form an Mb modified electrode. Mb can react di-
rectly on the modified electrode, and yield a pair of well-defined, quasi-reversible redox peaks with 0.304 V (vs. SCE) of E00 in pH 7.0 PBS, indicating that synergistic effects of DNA film and BPPF6 provided an excellent microenvironment to achieve the direct electrochemistry of Mb [25]. The Mb modified electrode showed excellent electrocatalytic activity toward H2O2 and TCA with low detection limits. The low KM values toward H2O2 and TCA further demonstrated that Mb retained its high bioactivity in the DNA film with the CILE as the basic electrode. So, the DNA/CILE had potential to study other proteins. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 20675062, 20875076) and the Research Foundation for the Doctoral Program of Higher Education of China (No. 20060697013). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
E. Lojou, P. Bianco, Electroanalysis 16 (2004) 1113. X.H. Kang, J. Wang, Z.W. Tang, H. Wu, Y.H. Lin, Talanta 78 (2009) 120. J.W. Wang, L.P. Wang, J.W. Di, Y.F. Tu, Talanta 77 (2009) 1454. K. Qiao, N.F. Hu, Bioelectrochemistry 75 (2009) 71. Q. Xu, X.J. Bian, L.L. Li, X.Y. Hu, M. Sun, D. Chen, Y. Wang, Electrochem. Commun. 10 (2008) 995. N. Li, H.W. Zhao, R. Yuan, K.F. Peng, Y.Q. Chai, Electrochim. Acta 54 (2008) 235. C.H. Fan, G.X. Li, J.Q. Zhu, D.X. Zhu, Anal. Chim. Acta 423 (2000) 95. G. Wang, J.J. Xu, H.Y. Chen, Electrochem. Commun. 4 (2002) 506. P. Du, S.N. Liu, P. Wu, C.X. Cai, Electrochim. Acta 52 (2007) 6534. X.B. Lu, J.Q. Hu, X. Yao, Z.P. Wang, J.H. Li, Biomacromolecules 7 (2006) 975. Y. Liu, M. Wang, J. Li, P. He, H.T. Liu, J.H. Li, Chem. Commun. (2005) 1778. N. Maleki, A. Safavi, F. Tajabadi, Anal. Chem. 78 (2006) 3820. W. Sun, R.F. Gao, R.F. Bi, K. Jiao, Chinese J. Anal. Chem. 4 (2007) 567. W. Sun, R.F. Gao, K. Jiao, J. Phys. Chem. B 111 (2007) 4560. W. Sun, Z.Q. Zhai, D.D. Wang, S.F. Liu, K. Jiao, Bioelectrochemistry 74 (2009) 295. W. Sun, R.F. Gao, X.Q. Li, D.D. Wang, M.X. Yang, K. Jiao, Electroanalysis 20 (10) (2008) 1048. R.T. Kachoosangi, G.G. Wildgoose, R.G. Compton, Electroanalysis 19 (2007) 1483. E. Laviron, J. Electroanal. Chem. 101 (1979) 19. Y.G. Liu, W.H. Hou, C.L. Lu, J.J. Zhu, Anal. Biochem. 375 (2008) 27. R.A. Kamin, G.S. Wilson, Anal. Chem. 52 (1980) 1198. C.Y. Liu, J.M. Hu, Biosens. Bioelectron. 24 (2009) 2149. H.J. Chen, S.J. Dong, Biosens. Bioelectron. 22 (2007) 1811. H.Y. Ma, N.F. Hu, J.F. Rusling, Langmuir 16 (2000) 4969. W. Sun, X.Q. Li, Y. Wang, R.J. Zhao, K. Jiao, Electrochim. Acta 54 (2009) 4141. J.S. Long, D.S. Silvester, G.G. Wildgoose, A.-E. Surkus, G.-U. Flechsig, R.G. Compton, Bioelectrochemistry 74 (2008) 183.
Contents lists available at ScienceDirect
Electrochemistry Communications journal homepage: www.elsevier.com/locate/elecom
Direct electrochemistry of myoglobin based on DNA accumulation on carbon ionic liquid electrode Ruifang Gao, Jianbin Zheng * Institute of Analytical Science, Shaanxi Provincial Key Laboratory of Electroanalytical Chemistry, Northwest University, Xi’an, Shaanxi 710069, PR China
a r t i c l e
i n f o
Article history: Received 5 May 2009 Received in revised form 21 May 2009 Accepted 22 May 2009 Available online 29 May 2009 Keywords: Electroanalysis Direct electrochemistry Electrostatic interaction Ionic liquid Myoglobin Deoxyribonucleic acid
a b s t r a c t Poly-anionic deoxyribonucleic acid (DNA) was accumulated on the positively charged surface of carbon ionic liquid electrode (CILE) with N-butylpyridinium hexafluorophosphate (BPPF6) as binder, and then myoglobin (Mb) was immobilized onto the DNA film by electrostatic interaction to form Mb/DNA/CILE electrode. The direct electrochemistry of Mb was then investigated in detail. A pair of well-defined, quasi-reversible cyclic voltammetric peaks of Mb was obtained with the formal potentials (E00 ) at 0.304 V (vs. SCE) in phosphate buffer solution (PBS, pH 7.0). The Mb/DNA/CILE electrode showed excellent electrocatalytic activity to H2O2 and trichloroacetic acid (TCA) in the range of 1.0–160 lmol/L and 0.5–40.0 mmol/L, respectively. The apparent Michaelis–Menten constants (KM) toward H2O2 and TCA were calculated as 0.42 and 0.82 mmol/L. So, the DNA/CILE had potential to study other proteins. Ó 2009 Published by Elsevier B.V.
1. Introduction Different materials for immobilization enzymes on the surface of working electrode have been employed including sol-gel, polymers, nanoparticles [1–5]. Among them, deoxyribonucleic acid (DNA) as an important biological macromolecule has electrical conducting electrochemical properties and unique three dimensional structures. Immobilized DNA on the surface of a substrate electrode not only provides a biocompatible microenvironment for protein, but also dramatically increases the coverage of protein on the electrode surface [6]. So, more and more attention was paid on the application of DNA to biosensors. Fan et al. [7] adopted cast method to immobilize hemoglobin and DNA on pyrolytic graphite electrode and a nitric oxide biosensor was fabricated. Wang et al. [8] successfully immobilized multi-walled carbon nanotube on the surface of platinum electrode by mixing with DNA, and then cytochrome c was adsorbed onto the modified electrode to investigate its direct electrochemistry. Recently, room temperature ionic liquids (RTILs) have been employed as a new media in electrochemistry and electroanalysis. As green solvent with many specific physicochemical properties such as high ionic conductivity, wide electrochemical range, negligible vapor pressure and thermal stability, RTILs have been a promising choice not only for supporting electrolyte but also for the modified materials incorporating with conventional biocomposite matrixes * Corresponding author. Tel.: +86 29 88302077; fax: +86 29 88303448. E-mail address: [email protected] (J. Zheng). 1388-2481/$ - see front matter Ó 2009 Published by Elsevier B.V. doi:10.1016/j.elecom.2009.05.046
such as carbon materials, cellulose, chitosan, silica sol-gel [9–11] to investigate direct electrochemistry of redox proteins. RTILs as a binder were also widely used in biosensors. Maleki et al. [12] fabricated a high-performance carbon composite electrode (CILE) by using graphite powder mixed with n-octylpyridinium hexafluorophosphate as binder, the resulted CILE showed attractive electrochemical behaviors. Sun et al. [13] also combined N-butylpyridinium hexafluorophosphate (BPPF6) with graphite powder to make a new kind of CILE. Then, they successfully fabricated several protein biosensors layer-by-layer method with this CILE [14,15]. The aim of this work was to preparation a controllable thin DNA film on the surface of CILE (DNA/CILE) by electrochemistry method. Then, Mb was immobilized on DNA/CILE to investigate its behavior of direct electrochemistry on the DNA film. 2. Experimental 2.1. Reagents Bovine myoglobin (Mb, MW. 64500) and Salmon sperm dsDNA were from Sigma. N-butylpyridinium hexafluorophosphate (BPPF6, 97%) was purchased from Hangzhou Kemer Chemical Limited Company. Graphite powder (SP) was got from Sinopharm Chemical Reagent Co. Ltd. H2O2 (w/w, 30%) was got from Tianjin Tianda Chemistry Reagent Plant. TCA was from Shanghai Shanpu chemical plant Co. Ltd. 0.1 mol/L phosphate buffer solution (PBS) was used as the supporting electrolyte. Other reagents were analytical
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reagent grade and doubly distilled water was used in all the experiments. 2.2. Apparatus All the electrochemical experiments were carried out on a CHI660A electrochemical workstation (Shanghai CH Instrument Co. Ltd., China) using a three electrode system. The working electrode was made by the following procedure. A saturated calomel electrode (SCE) and a platinum electrode were served as reference and counter electrode, respectively. All the tested solutions were purged with highly purified nitrogen for 30 min before the experiments and a nitrogen environment was kept over the solution during the electrochemical measurements. 2.3. Preparation of Mb modified electrode CILE was prepared under the optimum condition: 1.2 g of graphite powder and 1.0 g of BPPF6 were mixed thoroughly in a mortar and heated at 80 °C for about 2 h to form a homogenous carbon paste. Then, a glass tube (U = 3 mm) and a copper wire were applied to fabricate the CILE. After carefully polishing the surface of CILE on a piece of weighting paper, the following procedure was performed to fabricate an Mb/DNA/CILE electrode. Firstly, a bare CILE was immersed in pH 7.0 PBS solution containing 2.0 mg/mL DNA at the potential of +1.5 V for 360 s. Then, the obtained DNA/CILE electrode was incubated in 10 mg/mL Mb solution at pH 5.0 for 10 h to fabricate the Mb/DNA/CILE electrode. DNA/ CILE and Mb/CILE were prepared in the same way.
3.2. Direct electrochemistry of Mb The CVs of the DNA/CILE, Mb/CILE and Mb/DNA/CILE modified electrodes were measured in 0.1 M PBS (pH 7.0) at 100 mV/s (Fig. 2). At DNA/CILE (curve a), no peak was found and a high background current was observed, which was due to the high capacitance of RTIL and it was the typical characteristic of CILE [12,16,17]. Curve b was the voltammogram of Mb/CILE, a very small redox peaks was appeared. After DNA accumulating on CILE as the film to adsorb Mb, a remarkable enhanced electrochemical response was observed (curve c). A pair of well-defined, quasireversible redox peaks of Mb were located at 0.336 and 0.271 V with the DEp of 65 mV and formal potential (E00 = (Epa + Epc)/2) of 0.304 V (vs. SCE), respectively. The electron transfer reactivity of Mb was significantly improved on the DNA film. It may be attribute to that the immobilization of DNA on electrode surface can form a conductive thin layer of nano-structures to further enhance the electrode surface area for immobilizing more Mb [6]. Cyclic voltammetry was applied to investigate the behaviors Mb/DNA/CILE in pH 7.0 PBS at different scan rate. Both the cathodic and anodic peak currents of Mb increased linearly with scan rates from 0.05 to 0.6 V/s, indicating that a typical surface controlled electrochemical process was achieved on Mb/DNA/CILE [14]. The anodic and cathodic peak potentials were linearly dependent on the logarithm of the scan rates with slopes of 2.3RT/anF and 2.3RT/(1a)nF [14]. So, the charge-transfer coefficient (a) was calculated as 0.57 and the number of electron transferred was 0.94 1, respectively. According to the equation model of Laviron [18], the electron transfer rate constant (ks) was further estimated to be 1.02 s1.
3. Results and discussion 3.3. Electrocatalytic behaviors to H2O2 and TCA 3.1. Characterization of DNA film The electrochemical behaviors of different modified electrodes 3=4 solution containing 0.1 M KCl were charactered by FeðCNÞ6 and the cyclic voltammograms (CVs) were shown in Fig. 1. At bare CILE (curve a), a reversible electrochemical response for FeðCNÞ63=4 was observed with a peak-to-peak separation (DEp) of 66 mV at 100 mV/s. After accumulation DNA on the bare CILE, both the cathodic and anodic peak currents decreased obviously and the DEp increased to 70 mV (curve b). It indicated that the DNA immobilized on the electrode had slowed down the electron transfer of the redox probe due to the electrostatic repulsion between the poly-anionic DNA and the redox couple ions carrying negative charge [6]. When Mb was immobilized on DNA/CILE, a further decrease of the peak currents of FeðCNÞ63=4 was obtained with DEp of 77 mV (curve c), which was due to the non-conductive properties of Mb that acted as an inert electron layer and hindered the electron transfer.
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The electrocatalytic reduction of H2O2 and TCA was studied by cyclic voltammetry. The catalytic reduction of H2O2 on Mb/DNA/ CILE electrode was first examined. As shown in Fig. 3A, after adding H2O2 into the deoxygenated pH 7.0 PBS, a new reduction peak appeared at about 0.368 V (vs. SCE). While, a reduction peak potential more than 0.7 V was obtained on the DNA/CILE with the addition of H2O2 (curve b). Thus, the reduction potential of H2O2 decreased about 0.4 V, indicating the presence of Mb in the DNA film decreased the reaction activation energy observably. With increasing the concentration of H2O2, the reduction peak current of Mb increased dramatically, while the anodic peak current decreased to almost zero (curve d–n), which demonstrated the typical electrocatalytic reduction process of H2O2 [19]. The reduction peak current had a linear relationship with the concentration of H2O2 in the range from 1.0 to 160 lmol/L. The linear regression equation was Ipc (lA) = 0.711 C (lmol/L) + 11.49 (n = 34, r = 0.9975) with a detection of 0.2 lmol/L (S/N = 3).
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Fig. 1. CVs obtained at (a) bare CILE, (b) DNA/CILE and (c) Mb/DNA/CILE in 1 mmol/ solution containing 0.1 mol/L KCl. Scan rate: 100 mV/s. L FeðCNÞ3=4 6
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Fig. 2. CVs of (a) DNA/CILE, (b) Mb/CILE, (c) Mb/DNA/CILE in pH 7.0 PBS at scan rate of 100 mV/s.
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-0.2 -0.4 -0.6 -0.8 -1.0 E/V(vs.SCE)
Fig. 3. (A) CVs of DNA/CILE in the presence of (a) 0, (b) 100.0 lmol/L H2O2, (c–n) Mb/DNA/CILE in the presence of 0, 8.0, 15.0, 20.0, 30.0, 40.0, 50.0, 60.0, 70.0, 80.0, 90.0, 100.0, 120.0 lmol/L H2O2, respectively. Inset: the linear part of the calibration curve. (B) CVs of DNA/CILE in the presence of (a) 0, (b) 2.0 mmol/L TCA, (c–l) Mb/DNA/CILE in the presence of 0, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0 mmol/L TCA, respectively. Inset: relationship of the catalytic peak current and the concentration of TCA. Scan rate: 100 mV/s.
When the concentration of H2O2 was more than 1.6 104 mol/L, a response plateau was observed, showing a typical Michaelis–Menten kinetic mechanism. The enzyme-substrate kinetics could be indicated by the apparent Michaelis–Menten constant (KM), which can be obtained from the electrochemical version of Lineweaver–Burk equation [20]. So, the KM value was calculated as 0.42 mmol/L, which was lower than some previous reports [21,22], suggesting that Mb retained higher activity in the DNA film on the surface of CILE. Electrochemical catalytic reduction of TCA on the Mb/DNA/CILE was also investigated (Fig. 3B). After the addition of TCA into a pH 7.0 PBS, the reduction peak increased with the decrease of the oxidation peak significantly. The more the TCA was added, the greater the reduction peak current was increased (curve d–l). The direct reduction of TCA at DNA/CILE was not found in the potential range of 0.1 to 1.0 V. This indicated that the presence of Mb could reduce the over potential of TCA. The reduction peak current is linearly proportional to the concentration of TCA in the range of 0.5– 40.0 mmol/L (Fig. 3B inset) with the linear regression as Ipc(lA) = 1.06 C (104 mol/L) + 9.46 (n = 18, r = 0.9973) and the detection limit was 8.3 105 mol/L. In addition, according to the electrochemical version of Lineweaver–Burk equation [20], the KM was calculated as 0.82 mmol/L, which was lower than some previous reports [23,24], showing a high affinity to TCA. 3.4. Reproducibility and stability of the Mb/DNA/CILE The Mb modified electrode was used for 11 parallel determinations of 50.0 lmol/L H2O2 solution and the relative standard deviation (RSD) was got as 4.2%. The stability of the Mb modified electrode was evaluated by 80 continuous cyclic scans. No obvious decrease of the voltammetric response was found. After storing two weeks at 4 °C refrigerator, the modified electrode nearly remained its initial current response. After one-month storage, the modified electrode retained about 91.6% of the initial current response. The results indicated that DNA film was efficient for Mb immobilization and the bioactivity of Mb was remained. 4. Conclusions Poly-anionic DNA was accumulated on the positively charged surface of CILE electrode to form a conductive thin layer film of DNA, and then Mb was immobilized onto the film by electrostatic interaction to form an Mb modified electrode. Mb can react di-
rectly on the modified electrode, and yield a pair of well-defined, quasi-reversible redox peaks with 0.304 V (vs. SCE) of E00 in pH 7.0 PBS, indicating that synergistic effects of DNA film and BPPF6 provided an excellent microenvironment to achieve the direct electrochemistry of Mb [25]. The Mb modified electrode showed excellent electrocatalytic activity toward H2O2 and TCA with low detection limits. The low KM values toward H2O2 and TCA further demonstrated that Mb retained its high bioactivity in the DNA film with the CILE as the basic electrode. So, the DNA/CILE had potential to study other proteins. Acknowledgments This work was supported by the National Natural Science Foundation of China (Nos. 20675062, 20875076) and the Research Foundation for the Doctoral Program of Higher Education of China (No. 20060697013). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25]
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