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Electrochemical oxidation of histidine at an anodic oxidized boron-doped diamond electrode in neutral solution
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Electrochimica Acta 53 (2008) 2883–2889
Electrochemical oxidation of histidine at an anodic oxidized boron-doped diamond electrode in neutral solution Li-Chia Chen a , Chia-Chin Chang b,∗∗ , Hsien-Chang Chang a,c,d,∗ a
Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan 701 Department of Environment and Energy, National University of Tainan, Tainan, Taiwan 701 c Institute of Nanotechnology and Microsystems Engineering, National Cheng Kung University, Tainan, Taiwan 701 d Center for Micro/Nano Technology Research, National Cheng Kung University, Tainan, Taiwan 701 b
Received 13 August 2007; received in revised form 22 October 2007; accepted 29 October 2007 Available online 6 November 2007
Abstract Electrochemical oxidation of histidine (His) at an anodic oxidized boron-doped diamond electrode (AOBDDE) was performed. A significant peak of His oxidation is observed at about +1.5 V vs. Ag/AgCl, however, the response current was inhibited due to strong His-oxidized product adsorption onto the electrode surface. The characteristics of the His-oxidized product adsorbed onto the electrode surface were investigated by studying the electrochemical behavior of the Fe(CN)6 4− redox reaction using cycle voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Both CV and EIS results showed a decrease in the sum of transfer coefficients and an increase in the electron transfer resistance, which indicate that the adsorption film is a non-conductive film. The most possible active site locations for the AOBDDE for His oxidation are within these low-lying polycrystallite AOBDDE surface regions. The results from Raman and X-ray photoelectron spectroscopy offer strong evidence of the imidazole ring reaction from His. Experiments confirmed that the adsorbed film can be removed and the electrode surface reactivated using brief polarization at +2.5 V. © 2007 Published by Elsevier Ltd. Keywords: Histidine (His); Anodic oxidized boron-doped diamond; Electrochemical impedance spectroscopy; Raman; X-ray photoelectron spectroscopy
1. Introduction Histidine (His) is one of the necessary amino acids existing widely in muscular and nervous tissue. His constitutes the active center of many enzymes and brain nervous peptide and controls the transmission of metal elements in biological bases [1,2]. Based on electron transfer communication, poly-His with different metals as the active center for electrode modification to mimic biological reactions has been utilized for oxygen reduction [3–5]. The main intermediates and products [6–8], such as histamine, imidazole acetic acid and methyl imidazole acetic acid, play important roles in the metabolism of His. For example, histamine, one of the products, is a major factor that causes aller∗ Corresponding author at: Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan. Tel.: +88 662757575x63426; fax: +88 662760697. ∗∗ Corresponding author. Tel.: +88 662606123x7208; fax: +88 662602205. E-mail addresses: [email protected] (C.-C. Chang), [email protected] (H.-C. Chang).
0013-4686/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.electacta.2007.10.071
genic reactions. The electrochemical behavior of His becomes necessary for advanced investigation. However, little attention has been paid to the electrochemical reaction of His because it is electrochemically inactive in water. The adsorption and electrochemical reaction mechanism of His has been reported [9,10], requiring efficient and environmental friendly anode materials. Recently, boron-doped diamond (BDD) thin films have been used as a promising electrode material to detect electro-active species oxidation at high positive potentials, because of its specific physical and chemical properties such as hardness, chemical inertness, thermal conductivity and electrical conductivity [11,12]. BDD electrodes show a wide potential window in aqueous electrolytes (about −1.35 to 2.3 V/NHE) with low and stable voltametric background current density, permitting the detection of species that have been masked by the electrochemical decomposition of the solvent used or by surface reactions on classic carbon electrodes. Relative to other materials, the superiority of BDD has attracted considerable interest in various fields, including electroanalytical applications on different biomolecules [13–21]. Although the electrochemical reactions
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of some amino acids, including glycine, cysteine, tryptophan, and tyrosine, at the BDD electrode have been reported [14,22], there are relative few reports on His. The purpose of this work is to study the electrochemical behavior of His at an anodic oxidized BDD electrode. The adsorbed film characteristics of His-oxidized product was identified by studying the electrochemical behavior of the redox probe (Fe(CN)6 4− ) using cyclic voltammetry and electrochemical impedance spectroscopy. The scanning electron microscopy, Raman and X-ray photoelectron spectroscopy measurements were used to examine the His-oxidized product. Fast and simple removal of the adsorbed film using anodic polarization is also confirmed. 2. Experimental l-Histidine (His) was purchased from Sigma and used without further purification. The electrolyte is 0.5 M K2 SO4 (Merck) solution (pH 6.8). All solutions were prepared with pure water purified through a Millipore ultra-purification system. All other chemicals were reagent grade and used without further purification. All experiments were performed at 25 o C. This temperature was controlled (accuracy of 0.05 o C) using a water thermostat (HAAKE D8 and G). The as-deposited BDD electrodes, grown using chemical vapor deposition on one side of a silicon substrate material (Diachem Condias, Germany), were electrochemically oxidized in a 0.5 M K2 SO4 solution at +2.7 V for 30 min. The anodic oxidized BDD electrode (AOBDDE) was then housed in a Teflon mounting to allow contact with the electrolyte solution to the diamond thin film. The electrode was rinsed with de-ionized water prior to each voltammetric scan. Cyclic voltammetry was performed with an Autolab PGSTAT30 (ECO Chemie, B.V., Netherlands) and a three-electrode cell with an Ag/AgCl electrode acting as the reference and a Pt wire as the counter electrode. Electrochemical impedance spectroscopy was performed in the batch reactor by coupling the potentiostat with an Autolab frequency response analyzer locked in an amplifier and an impedance phase analyzer. Sinusoidal amplitude modulation of ±10 mV was used over a frequency range from 0.01 Hz to 100 kHz, with the potentials were operated at 0.25 V. For product adsorption, the AOBDDE was subjected to 1 h of polarization in 0.5 M K2 SO4 with 2 mM His at +1.5 V to induce a substantial adsorbed film sample. The surface morphology was investigated using a scanning electron microscope (JEOL JSM 35) operated at 20 keV. A micro-Raman scattering study of the electrode was performed using a confocal RENISHAW micro-Raman system (RENISHAW System 1000, Renishaw plc., Gloucestershire, U.K.) with the spectrometer equipped with 1800-lines/mm grating. Raman spectra recorded from the diamond top surface using a 20× objective to focus and adjust the scattered laser light. The excitation source was a 633 nm helium neon laser with a power of 17 mW. Data was calibrated with the silicon band at 520 cm−1 . Raman spectra were collected using a 100 s exposure time. All Raman spectral data were processed with software supplied by RENISHAW, and background corrections made as needed. X-ray photoelectron measurements
(VG. ESCA210 Electron Spectroscopy for Chemical Analysis) were used to characterize the chemical changes on the diamond surface with a non-monochromatic Al K␣ as X-ray source. Gaussian functions and a least-square routine were used to fit the peaks. 3. Results and discussion 3.1. Electrochemical reaction of histidine (His) at anodic oxidized boron-doped diamond electrode (AOBDDE) Fig. 1 shows the cyclic voltammograms of the AOBDDE in 0.5 M K2 SO4 solution with and without 2 mM His. An examination of Fig. 1(a) indicates that a significant peak can be observed in the first scan cycle at about 1.5 V after the addition of 2 mM His, which is attributed to the oxidation of His at the AOBDDE. Two amino acids with different residual groups, glycine and glutamic acid, were chosen to compare the electrochemical properties with His. Interestingly, no discernible oxidation peak was found for both glycine and glutamic acid in the same experimental condition shown in Fig. 1(a) (data not shown). Therefore, these results imply that the oxidation signal of His at AOBDDE might be contributed from the reaction of the imidazole ring of His. It also can be found in Fig. 1 that the peak current intensity decreases and the initial potential of the rising current shifts towards positive values with increasing scan cycles. No discernible His oxidation peak can be observed after a scan for 15 cycles, and the electrode surface remained inactive even after it was washed using de-ionized water and acetone. In previous research, the decrease in oxidation peak current intensity, because of the product deposition, caused by benzyl alcohol oxidation was reported [21]. Similarly, the inhibition of continuous His oxidation in this work was caused by the adsorption of His-oxidized product. 3.2. Characteristic of the product adsorbed film at AOBDDE To evaluate the product adsorbed film characteristics, the AOBDDE was pretreated with 1.5 V polarization in 0.5 M
Fig. 1. Cyclic voltammograms of AOBDDE in 0.5 M K2 SO4 solution in the absence (1) and the presence of 2 mM His for the 1st (2), 2nd (3), and 15th (4) cycle at a scan rate of 0.1 V s−1 .
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Fig. 3. (a) Nyquist and (b) Bode plots of AOBDDE without (♦), with () 2 mM, and () 4 mM His-oxidized treatment in 0.5 M M K2 SO4 solution with 4 mM Fe(CN)6 4− at 0.25 V.
with and without His-oxidized treatment are both proportional to the scan rates, suggesting a typical surface-controlled electrode process. Noteworthy is the dependence of the anodic (Ep, a) and cathodic (Ep, c) peak potentials on the potential sweep rate v, as shown in Fig. 2(c). Both AOBDDEs with and without Hisoxidized treatment show linear relationships. The anodic (αa ) and cathodic (αc ) transfer coefficient values can be determined Fig. 2. Cyclic voltammograms of AOBDDE without (a) and with (b) Hisoxidized treatment immersed in 0.5 M K2 SO4 solution with 4 mM Fe(CN)6 4− at different scan rate: (1) 0.01, (2) 0.02, (3) 0.03, (4) 0.04, (5) 0.05, (6) 0.1, (7) 0.2, and (8) 0.3 V s−1 and (c) dependence of peak potentials with the scan rate v ((♦), ( )for AOBDDE; (), (䊉) for AOBDDE with His-oxidized treatment).
K2 SO4 with different His concentrations for 600 s. The electrochemical characteristic of AOBDDE with and without His-oxidized treatment was measured in 0.5 M K2 SO4 with 4 mM Fe(CN)6 4− . The cyclic voltammograms of AOBDDE in 0.5 M K2 SO4 with 4 mM Fe(CN)6 4− at different scan rates show well-defined but irreversible redox peaks (Fig. 2(a)). However, 2 mM His-oxidized treatment results in larger redox peaks separation potential and inhibition of the peak current, as shown in Fig. 2(b). This means that a greater energy barrier for the Fe(CN)6 4− redox reaction at the His-oxidized treated AOBDDE is required. With the increasing scan rate ranging from 0.01 to 0.3 V s−1 , the AOBDDE anodic and cathodic peak currents
Fig. 4. Equivalent circuits for AOBDDE without (a) and with (b) His-oxidized treatment.
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Table 1 Parameters used for fitting the impedance results in Fig. 3 for the electrodes without and with His-oxidized treatment, based on the equivalent circuit in Fig. 4 Diamond sample
Rs ()
RCT1 (k)
CPE1 (F)
RCT2 (k)
CPE2 (F)
Initial 2 mM His-oxidized treatment 4 mM His-oxidized treatment
34.3 33.0 33.2
2.1 13.35 14.76
1.1 1.0 1.1
– 17.43 21.83
– 148 117
using the formula [23]: RT Ep = consant − ln v 2αnF
(1)
where n = 1 is the number of electrons transferred per molecule of the redox probe, R = 8.31 J mol−1 K−1 the gas constant, T = 298 K the experimental temperature, F = 96500 C the Faraday constant, and v is the scan rate. The anode and cathode transfer coefficients from AOBDDE are equal to 0.37 and 0.26, respectively. However, they become equal to 0.30 and 0.22, respectively, from the AOBDDE with His-oxidized treatment. His oxidation at AOBDDE results in a change in the transfer coefficients sum from 0.63 to 0.52, indicating the decrease in electrochemical activity. Electrochemical impedance spectrograms of AOBDDE with and without His-oxidized treatment are shown in Fig. 3. The difference in Nyquist and Bode plots caused by His-oxidized treatment indicates a variation of AOBDDE electrochemical
Fig. 5. SEM images of (a) AOBDDE and (b) the same electrode in 0.5 M K2 SO4 with 2 mM His at 1.5 V for 1 h polarization.
characteristics. The AOBDDE Nyquist plot displays a single semicircular loop followed by a 45◦ straight line. This plot becomes two semicircular loops after His-oxidized treatment. The curve has two significant semicircular loops observed in the Bode plots (phase vs. log f) of His-oxidized treatment AOBDDE. All of these observations infer the formation of a new interface, the product deposition, between the electrode and solution after His oxidation. The AOBDDE electrochemical characteristics can be described by fitting the impedance data to a simple Randles equivalent circuit. However, two RC time constants in the equivalent circuit modeling are fitted for the AOBDDE with His-oxidized treatment, as shown in Fig. 4. The fitting parameters are summarized in Table 1. The increasing resistance in the electrode/adsorbed product/solution interface implies increasing charge transfer inhibition for the redox active species observed for the AOBDDE with His-oxidized treatment. Therefore, it can be confirmed that His oxidation at AOBDDE causes the adsorption of a non-conductive film at the electrode surface.
Fig. 6. Raman spectra of (a) AOBDDE and (b) the same electrode in 0.5 M K2 SO4 with 2 mM His at 1.5 V for 1 h polarization.
L.-C. Chen et al. / Electrochimica Acta 53 (2008) 2883–2889
Fig. 7. XPS narrow scans of AOBDDE (A, B, and C) and the same electrode in 0.5 M K2 SO4 with 2 mM His at 1.5 V for 1 h polarization (a, b, and c).
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3.3. SEM, Raman and XPS measurements AOBDDE was immersed in 0.5 M K2 SO4 solution with 2 mM His at +1.5 V for 1 h to form a significant film for subsequent measurements. Clearly defined twin crystallites with distinct faceting and random orientation can be observed in the typical SEM image of AOBDDE, as shown in Fig. 5(a). Other materials located at the low-lying regions between the diamond crystallites in Fig. 5(b) suggest oxidized product deposition. Based on the SEM images, the most probable oxidized product deposition situation is considered as adsorption at specific sites on the BDD electrode surface. Furthermore, these specific sites are the active BDD electrode sites. In Fig. 6(a), a sharp Raman signal peak approximately at 1334 cm−1 is the well-known crystalline diamond characteristic [24]. The other broad peak at 1200 cm−1 is possibly Fano-type asymmetry of the 1334 cm−1 peak or, alternatively, may be
files show no significant difference between the AOBDDEs with and without His-oxidized treatment. The nitrogen profile signals from the AOBDDE with His-oxidized treatment are identified as C–N, C N, and C–N+ , respectively. Although the accurate reaction mechanism is still not clear, the above results can confirm the residual group reaction for His oxidation in this work. The His reaction at AOBBDE is considered to form adsorbed film through polymerization, which occurs at the C C bond of the imidazole ring. In previous reports [13,14,16–21], the generation of powerful oxidants, such as OH radicals and S2 O8 2− at AOBDDE affects the electrochemical reaction and causes indirect oxidation. Therefore, the polymerization reaction in this study is considered to involve powerful oxidants. The polymerization reaction mechanism is suggested as follows. However, other reaction mechanisms are also possible. −e−
BDD + H2 O/SO4 2− −→ · OH/S2 O8 2− + BDD
associated with photo-luminescence [24]. In comparison with Fig. 6(a), the additional Raman signals, shown in Fig. 6(b), are interpreted as attributed from the adsorbed product. The signals in the range from 1000 to 1200 cm−1 are attributed to C–N bond vibration of the imidazole ring from His [25]. The signal at about 1223 cm−1 is identified as C–H bond vibration [25]. Interestingly, there is no Raman signal for the C C vibration (∼1600 cm−1 ) contributed from the imidazole structure, which may suggest the imidazole reaction in this work. Fig. 7 shows the XPS results for the C1, N1 and O1 spectra regions of the AOBDDE with and without His-oxidized treatment. A peak fitting with Gaussian peaks to identify the different chemical states present was carried out. As shown in the carbon peak profile, the sp3 -carbon contents, centered at 284.4 eV are the most intense peaks in the AOBDDE component. The peaks at higher bonding energies are identified as corresponding to oxygen-bound carbons on the diamond surface (285.5 eV: C–O and 286.4 eV: C O) [26,27]. Comparatively, the C–N, C N (286.4 eV) and COOH (285.5 eV) peaks were also observed from the AOBDDE with His-oxidized treatment. However, the same as Raman result, there is no sp2 -carbon content signal (C C), centered at 283.3 eV [26]. The oxygen peak pro-
3.4. Electrode refresh As described above, a non-conductive film is deposited at AOBDDE after His oxidation. It is desirable to remove this film
Fig. 8. Cyclic voltammograms of AOBDDE in 0.5 M K2 SO4 solution with (1) 1, (2) 2, (3) 3, and (4) 4 mM His at a scan rate of 0.1 V s−1 . The AOBDDE is polarized at +2.5 V for 30 s after each scan of His oxidation. Inset figure: linear calibration plot of response peak current against His concentration.
L.-C. Chen et al. / Electrochimica Acta 53 (2008) 2883–2889
and refresh the electrode to retain electrode activity for prolonged use. As mentioned in the reports [19,21], OH radicals are generated at standard potential about 2.11 V (vs. Ag/AgCl), which can assist in removing these deposited materials. The electrode surface becomes reactive. Each cyclic voltammetry step for AOBDDE in different His concentrations was measured for 15 scan cycles. After that, the electrode was immersed in 0.5 M K2 SO4 at +2.5 V for 30 s polarization to refresh for the next measurement. Fig. 8 shows the cyclic voltammetry curves for the first cycle in each step. The inset figure shows linear behavior for the His concentration dependence on the peak current, which indicates that the intermittent +2.5 V treatment removed the deposited material, and the electrode retained its full electrochemical activity. 4. Conclusion In this study, a well-defined His oxidation signal can be found at about 1.5 V vs. Ag/AgCl in 0.5 M K2 SO4 at AOBDDE. Experiments confirmed the adsorption of a non-conductive film from His-oxidized product at the AOBDD electrode surface. This film reduces the electrode electrochemical activity. SEM images show the adsorbed materials clearly at the low-lying regions of the polycrystallite AOBDD surface, which are considered the most possible active sites of His oxidation. Raman spectrograms show the C–N bond vibration signal from the AOBDDE with His-oxidized treatment. XPS results offer evidence of C–N and COOH foundational groups for the AOBDDE with Hisoxidized treatment. However, no C C signals were exhibited in the Raman and XPS spectrograms. These results strongly suggest a reaction at the imidazole ring of His at the AOBDDE, but the His reaction mechanism in this work is still not clearly identified. An efficient and easy method (brief polarization) was used to remove the adsorbed film and make the electrode reactive. Acknowledgements The authors thank the Center for Micro/Nano Technology Research and the Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan, for equipment access and technical support.
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Electrochimica Acta 53 (2008) 2883–2889
Electrochemical oxidation of histidine at an anodic oxidized boron-doped diamond electrode in neutral solution Li-Chia Chen a , Chia-Chin Chang b,∗∗ , Hsien-Chang Chang a,c,d,∗ a
Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan 701 Department of Environment and Energy, National University of Tainan, Tainan, Taiwan 701 c Institute of Nanotechnology and Microsystems Engineering, National Cheng Kung University, Tainan, Taiwan 701 d Center for Micro/Nano Technology Research, National Cheng Kung University, Tainan, Taiwan 701 b
Received 13 August 2007; received in revised form 22 October 2007; accepted 29 October 2007 Available online 6 November 2007
Abstract Electrochemical oxidation of histidine (His) at an anodic oxidized boron-doped diamond electrode (AOBDDE) was performed. A significant peak of His oxidation is observed at about +1.5 V vs. Ag/AgCl, however, the response current was inhibited due to strong His-oxidized product adsorption onto the electrode surface. The characteristics of the His-oxidized product adsorbed onto the electrode surface were investigated by studying the electrochemical behavior of the Fe(CN)6 4− redox reaction using cycle voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Both CV and EIS results showed a decrease in the sum of transfer coefficients and an increase in the electron transfer resistance, which indicate that the adsorption film is a non-conductive film. The most possible active site locations for the AOBDDE for His oxidation are within these low-lying polycrystallite AOBDDE surface regions. The results from Raman and X-ray photoelectron spectroscopy offer strong evidence of the imidazole ring reaction from His. Experiments confirmed that the adsorbed film can be removed and the electrode surface reactivated using brief polarization at +2.5 V. © 2007 Published by Elsevier Ltd. Keywords: Histidine (His); Anodic oxidized boron-doped diamond; Electrochemical impedance spectroscopy; Raman; X-ray photoelectron spectroscopy
1. Introduction Histidine (His) is one of the necessary amino acids existing widely in muscular and nervous tissue. His constitutes the active center of many enzymes and brain nervous peptide and controls the transmission of metal elements in biological bases [1,2]. Based on electron transfer communication, poly-His with different metals as the active center for electrode modification to mimic biological reactions has been utilized for oxygen reduction [3–5]. The main intermediates and products [6–8], such as histamine, imidazole acetic acid and methyl imidazole acetic acid, play important roles in the metabolism of His. For example, histamine, one of the products, is a major factor that causes aller∗ Corresponding author at: Institute of Biomedical Engineering, National Cheng Kung University, Tainan, Taiwan. Tel.: +88 662757575x63426; fax: +88 662760697. ∗∗ Corresponding author. Tel.: +88 662606123x7208; fax: +88 662602205. E-mail addresses: [email protected] (C.-C. Chang), [email protected] (H.-C. Chang).
0013-4686/$ – see front matter © 2007 Published by Elsevier Ltd. doi:10.1016/j.electacta.2007.10.071
genic reactions. The electrochemical behavior of His becomes necessary for advanced investigation. However, little attention has been paid to the electrochemical reaction of His because it is electrochemically inactive in water. The adsorption and electrochemical reaction mechanism of His has been reported [9,10], requiring efficient and environmental friendly anode materials. Recently, boron-doped diamond (BDD) thin films have been used as a promising electrode material to detect electro-active species oxidation at high positive potentials, because of its specific physical and chemical properties such as hardness, chemical inertness, thermal conductivity and electrical conductivity [11,12]. BDD electrodes show a wide potential window in aqueous electrolytes (about −1.35 to 2.3 V/NHE) with low and stable voltametric background current density, permitting the detection of species that have been masked by the electrochemical decomposition of the solvent used or by surface reactions on classic carbon electrodes. Relative to other materials, the superiority of BDD has attracted considerable interest in various fields, including electroanalytical applications on different biomolecules [13–21]. Although the electrochemical reactions
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of some amino acids, including glycine, cysteine, tryptophan, and tyrosine, at the BDD electrode have been reported [14,22], there are relative few reports on His. The purpose of this work is to study the electrochemical behavior of His at an anodic oxidized BDD electrode. The adsorbed film characteristics of His-oxidized product was identified by studying the electrochemical behavior of the redox probe (Fe(CN)6 4− ) using cyclic voltammetry and electrochemical impedance spectroscopy. The scanning electron microscopy, Raman and X-ray photoelectron spectroscopy measurements were used to examine the His-oxidized product. Fast and simple removal of the adsorbed film using anodic polarization is also confirmed. 2. Experimental l-Histidine (His) was purchased from Sigma and used without further purification. The electrolyte is 0.5 M K2 SO4 (Merck) solution (pH 6.8). All solutions were prepared with pure water purified through a Millipore ultra-purification system. All other chemicals were reagent grade and used without further purification. All experiments were performed at 25 o C. This temperature was controlled (accuracy of 0.05 o C) using a water thermostat (HAAKE D8 and G). The as-deposited BDD electrodes, grown using chemical vapor deposition on one side of a silicon substrate material (Diachem Condias, Germany), were electrochemically oxidized in a 0.5 M K2 SO4 solution at +2.7 V for 30 min. The anodic oxidized BDD electrode (AOBDDE) was then housed in a Teflon mounting to allow contact with the electrolyte solution to the diamond thin film. The electrode was rinsed with de-ionized water prior to each voltammetric scan. Cyclic voltammetry was performed with an Autolab PGSTAT30 (ECO Chemie, B.V., Netherlands) and a three-electrode cell with an Ag/AgCl electrode acting as the reference and a Pt wire as the counter electrode. Electrochemical impedance spectroscopy was performed in the batch reactor by coupling the potentiostat with an Autolab frequency response analyzer locked in an amplifier and an impedance phase analyzer. Sinusoidal amplitude modulation of ±10 mV was used over a frequency range from 0.01 Hz to 100 kHz, with the potentials were operated at 0.25 V. For product adsorption, the AOBDDE was subjected to 1 h of polarization in 0.5 M K2 SO4 with 2 mM His at +1.5 V to induce a substantial adsorbed film sample. The surface morphology was investigated using a scanning electron microscope (JEOL JSM 35) operated at 20 keV. A micro-Raman scattering study of the electrode was performed using a confocal RENISHAW micro-Raman system (RENISHAW System 1000, Renishaw plc., Gloucestershire, U.K.) with the spectrometer equipped with 1800-lines/mm grating. Raman spectra recorded from the diamond top surface using a 20× objective to focus and adjust the scattered laser light. The excitation source was a 633 nm helium neon laser with a power of 17 mW. Data was calibrated with the silicon band at 520 cm−1 . Raman spectra were collected using a 100 s exposure time. All Raman spectral data were processed with software supplied by RENISHAW, and background corrections made as needed. X-ray photoelectron measurements
(VG. ESCA210 Electron Spectroscopy for Chemical Analysis) were used to characterize the chemical changes on the diamond surface with a non-monochromatic Al K␣ as X-ray source. Gaussian functions and a least-square routine were used to fit the peaks. 3. Results and discussion 3.1. Electrochemical reaction of histidine (His) at anodic oxidized boron-doped diamond electrode (AOBDDE) Fig. 1 shows the cyclic voltammograms of the AOBDDE in 0.5 M K2 SO4 solution with and without 2 mM His. An examination of Fig. 1(a) indicates that a significant peak can be observed in the first scan cycle at about 1.5 V after the addition of 2 mM His, which is attributed to the oxidation of His at the AOBDDE. Two amino acids with different residual groups, glycine and glutamic acid, were chosen to compare the electrochemical properties with His. Interestingly, no discernible oxidation peak was found for both glycine and glutamic acid in the same experimental condition shown in Fig. 1(a) (data not shown). Therefore, these results imply that the oxidation signal of His at AOBDDE might be contributed from the reaction of the imidazole ring of His. It also can be found in Fig. 1 that the peak current intensity decreases and the initial potential of the rising current shifts towards positive values with increasing scan cycles. No discernible His oxidation peak can be observed after a scan for 15 cycles, and the electrode surface remained inactive even after it was washed using de-ionized water and acetone. In previous research, the decrease in oxidation peak current intensity, because of the product deposition, caused by benzyl alcohol oxidation was reported [21]. Similarly, the inhibition of continuous His oxidation in this work was caused by the adsorption of His-oxidized product. 3.2. Characteristic of the product adsorbed film at AOBDDE To evaluate the product adsorbed film characteristics, the AOBDDE was pretreated with 1.5 V polarization in 0.5 M
Fig. 1. Cyclic voltammograms of AOBDDE in 0.5 M K2 SO4 solution in the absence (1) and the presence of 2 mM His for the 1st (2), 2nd (3), and 15th (4) cycle at a scan rate of 0.1 V s−1 .
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Fig. 3. (a) Nyquist and (b) Bode plots of AOBDDE without (♦), with () 2 mM, and () 4 mM His-oxidized treatment in 0.5 M M K2 SO4 solution with 4 mM Fe(CN)6 4− at 0.25 V.
with and without His-oxidized treatment are both proportional to the scan rates, suggesting a typical surface-controlled electrode process. Noteworthy is the dependence of the anodic (Ep, a) and cathodic (Ep, c) peak potentials on the potential sweep rate v, as shown in Fig. 2(c). Both AOBDDEs with and without Hisoxidized treatment show linear relationships. The anodic (αa ) and cathodic (αc ) transfer coefficient values can be determined Fig. 2. Cyclic voltammograms of AOBDDE without (a) and with (b) Hisoxidized treatment immersed in 0.5 M K2 SO4 solution with 4 mM Fe(CN)6 4− at different scan rate: (1) 0.01, (2) 0.02, (3) 0.03, (4) 0.04, (5) 0.05, (6) 0.1, (7) 0.2, and (8) 0.3 V s−1 and (c) dependence of peak potentials with the scan rate v ((♦), ( )for AOBDDE; (), (䊉) for AOBDDE with His-oxidized treatment).
K2 SO4 with different His concentrations for 600 s. The electrochemical characteristic of AOBDDE with and without His-oxidized treatment was measured in 0.5 M K2 SO4 with 4 mM Fe(CN)6 4− . The cyclic voltammograms of AOBDDE in 0.5 M K2 SO4 with 4 mM Fe(CN)6 4− at different scan rates show well-defined but irreversible redox peaks (Fig. 2(a)). However, 2 mM His-oxidized treatment results in larger redox peaks separation potential and inhibition of the peak current, as shown in Fig. 2(b). This means that a greater energy barrier for the Fe(CN)6 4− redox reaction at the His-oxidized treated AOBDDE is required. With the increasing scan rate ranging from 0.01 to 0.3 V s−1 , the AOBDDE anodic and cathodic peak currents
Fig. 4. Equivalent circuits for AOBDDE without (a) and with (b) His-oxidized treatment.
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Table 1 Parameters used for fitting the impedance results in Fig. 3 for the electrodes without and with His-oxidized treatment, based on the equivalent circuit in Fig. 4 Diamond sample
Rs ()
RCT1 (k)
CPE1 (F)
RCT2 (k)
CPE2 (F)
Initial 2 mM His-oxidized treatment 4 mM His-oxidized treatment
34.3 33.0 33.2
2.1 13.35 14.76
1.1 1.0 1.1
– 17.43 21.83
– 148 117
using the formula [23]: RT Ep = consant − ln v 2αnF
(1)
where n = 1 is the number of electrons transferred per molecule of the redox probe, R = 8.31 J mol−1 K−1 the gas constant, T = 298 K the experimental temperature, F = 96500 C the Faraday constant, and v is the scan rate. The anode and cathode transfer coefficients from AOBDDE are equal to 0.37 and 0.26, respectively. However, they become equal to 0.30 and 0.22, respectively, from the AOBDDE with His-oxidized treatment. His oxidation at AOBDDE results in a change in the transfer coefficients sum from 0.63 to 0.52, indicating the decrease in electrochemical activity. Electrochemical impedance spectrograms of AOBDDE with and without His-oxidized treatment are shown in Fig. 3. The difference in Nyquist and Bode plots caused by His-oxidized treatment indicates a variation of AOBDDE electrochemical
Fig. 5. SEM images of (a) AOBDDE and (b) the same electrode in 0.5 M K2 SO4 with 2 mM His at 1.5 V for 1 h polarization.
characteristics. The AOBDDE Nyquist plot displays a single semicircular loop followed by a 45◦ straight line. This plot becomes two semicircular loops after His-oxidized treatment. The curve has two significant semicircular loops observed in the Bode plots (phase vs. log f) of His-oxidized treatment AOBDDE. All of these observations infer the formation of a new interface, the product deposition, between the electrode and solution after His oxidation. The AOBDDE electrochemical characteristics can be described by fitting the impedance data to a simple Randles equivalent circuit. However, two RC time constants in the equivalent circuit modeling are fitted for the AOBDDE with His-oxidized treatment, as shown in Fig. 4. The fitting parameters are summarized in Table 1. The increasing resistance in the electrode/adsorbed product/solution interface implies increasing charge transfer inhibition for the redox active species observed for the AOBDDE with His-oxidized treatment. Therefore, it can be confirmed that His oxidation at AOBDDE causes the adsorption of a non-conductive film at the electrode surface.
Fig. 6. Raman spectra of (a) AOBDDE and (b) the same electrode in 0.5 M K2 SO4 with 2 mM His at 1.5 V for 1 h polarization.
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Fig. 7. XPS narrow scans of AOBDDE (A, B, and C) and the same electrode in 0.5 M K2 SO4 with 2 mM His at 1.5 V for 1 h polarization (a, b, and c).
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3.3. SEM, Raman and XPS measurements AOBDDE was immersed in 0.5 M K2 SO4 solution with 2 mM His at +1.5 V for 1 h to form a significant film for subsequent measurements. Clearly defined twin crystallites with distinct faceting and random orientation can be observed in the typical SEM image of AOBDDE, as shown in Fig. 5(a). Other materials located at the low-lying regions between the diamond crystallites in Fig. 5(b) suggest oxidized product deposition. Based on the SEM images, the most probable oxidized product deposition situation is considered as adsorption at specific sites on the BDD electrode surface. Furthermore, these specific sites are the active BDD electrode sites. In Fig. 6(a), a sharp Raman signal peak approximately at 1334 cm−1 is the well-known crystalline diamond characteristic [24]. The other broad peak at 1200 cm−1 is possibly Fano-type asymmetry of the 1334 cm−1 peak or, alternatively, may be
files show no significant difference between the AOBDDEs with and without His-oxidized treatment. The nitrogen profile signals from the AOBDDE with His-oxidized treatment are identified as C–N, C N, and C–N+ , respectively. Although the accurate reaction mechanism is still not clear, the above results can confirm the residual group reaction for His oxidation in this work. The His reaction at AOBBDE is considered to form adsorbed film through polymerization, which occurs at the C C bond of the imidazole ring. In previous reports [13,14,16–21], the generation of powerful oxidants, such as OH radicals and S2 O8 2− at AOBDDE affects the electrochemical reaction and causes indirect oxidation. Therefore, the polymerization reaction in this study is considered to involve powerful oxidants. The polymerization reaction mechanism is suggested as follows. However, other reaction mechanisms are also possible. −e−
BDD + H2 O/SO4 2− −→ · OH/S2 O8 2− + BDD
associated with photo-luminescence [24]. In comparison with Fig. 6(a), the additional Raman signals, shown in Fig. 6(b), are interpreted as attributed from the adsorbed product. The signals in the range from 1000 to 1200 cm−1 are attributed to C–N bond vibration of the imidazole ring from His [25]. The signal at about 1223 cm−1 is identified as C–H bond vibration [25]. Interestingly, there is no Raman signal for the C C vibration (∼1600 cm−1 ) contributed from the imidazole structure, which may suggest the imidazole reaction in this work. Fig. 7 shows the XPS results for the C1, N1 and O1 spectra regions of the AOBDDE with and without His-oxidized treatment. A peak fitting with Gaussian peaks to identify the different chemical states present was carried out. As shown in the carbon peak profile, the sp3 -carbon contents, centered at 284.4 eV are the most intense peaks in the AOBDDE component. The peaks at higher bonding energies are identified as corresponding to oxygen-bound carbons on the diamond surface (285.5 eV: C–O and 286.4 eV: C O) [26,27]. Comparatively, the C–N, C N (286.4 eV) and COOH (285.5 eV) peaks were also observed from the AOBDDE with His-oxidized treatment. However, the same as Raman result, there is no sp2 -carbon content signal (C C), centered at 283.3 eV [26]. The oxygen peak pro-
3.4. Electrode refresh As described above, a non-conductive film is deposited at AOBDDE after His oxidation. It is desirable to remove this film
Fig. 8. Cyclic voltammograms of AOBDDE in 0.5 M K2 SO4 solution with (1) 1, (2) 2, (3) 3, and (4) 4 mM His at a scan rate of 0.1 V s−1 . The AOBDDE is polarized at +2.5 V for 30 s after each scan of His oxidation. Inset figure: linear calibration plot of response peak current against His concentration.
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and refresh the electrode to retain electrode activity for prolonged use. As mentioned in the reports [19,21], OH radicals are generated at standard potential about 2.11 V (vs. Ag/AgCl), which can assist in removing these deposited materials. The electrode surface becomes reactive. Each cyclic voltammetry step for AOBDDE in different His concentrations was measured for 15 scan cycles. After that, the electrode was immersed in 0.5 M K2 SO4 at +2.5 V for 30 s polarization to refresh for the next measurement. Fig. 8 shows the cyclic voltammetry curves for the first cycle in each step. The inset figure shows linear behavior for the His concentration dependence on the peak current, which indicates that the intermittent +2.5 V treatment removed the deposited material, and the electrode retained its full electrochemical activity. 4. Conclusion In this study, a well-defined His oxidation signal can be found at about 1.5 V vs. Ag/AgCl in 0.5 M K2 SO4 at AOBDDE. Experiments confirmed the adsorption of a non-conductive film from His-oxidized product at the AOBDD electrode surface. This film reduces the electrode electrochemical activity. SEM images show the adsorbed materials clearly at the low-lying regions of the polycrystallite AOBDD surface, which are considered the most possible active sites of His oxidation. Raman spectrograms show the C–N bond vibration signal from the AOBDDE with His-oxidized treatment. XPS results offer evidence of C–N and COOH foundational groups for the AOBDDE with Hisoxidized treatment. However, no C C signals were exhibited in the Raman and XPS spectrograms. These results strongly suggest a reaction at the imidazole ring of His at the AOBDDE, but the His reaction mechanism in this work is still not clearly identified. An efficient and easy method (brief polarization) was used to remove the adsorbed film and make the electrode reactive. Acknowledgements The authors thank the Center for Micro/Nano Technology Research and the Center for Bioscience and Biotechnology, National Cheng Kung University, Tainan, Taiwan, for equipment access and technical support.
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