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Electropolymerized toluidine blue O functionalized ordered mesoporous carbon-ionic liquid gel-modified electrode and its low-potential detection of NADH
Sensors and Actuators B 178 (2013) 169–175
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electropolymerized toluidine blue O functionalized ordered mesoporous carbon-ionic liquid gel-modified electrode and its low-potential detection of NADH Xiurong Zhai a,∗ , Yonghong Li b , Guangjun Liu a , Yang Cao c , Hongtao Gao a , Chengyang Yue a , Ning Sheng a a
Department of Chemistry and Chemical Engineering, Jining University, Qufu 273155, PR China School of Public Health, Ningxia Medical University, Ningxia, Yinchuan 750004, PR China c Center of Total Pollutants Control, Jining Environmental Protection Bureau, Jining 272000, PR China b
a r t i c l e
i n f o
Article history: Received 9 July 2012 Received in revised form 12 December 2012 Accepted 17 December 2012 Available online 26 December 2012 Keywords: Toluidine blue O Ordered mesoporous carbon Ionic liquid Synergistic effect NADH
a b s t r a c t A novel gel was developed based on the polymerization of a composite containing toluidine blue O (TBO) functionalized ordered mesoporous carbon (OMC) by a – stacking interaction and ionic liquid (IL). The poly (toluidine blue O) (PTBO)-OMC-IL composite film distributes almost homogeneously with unique structure on the surface of substrate. The PTBO-OMC-IL gel modified glass carbon (GC) electrode displays high conductivity investigated by electrochemical method. Electrochemical studies suggest that the PTBO-OMC-IL gel/GC electrode provide a positively synergistic effect among PTBO, OMC and IL on the electrochemical oxidation of NADH. Under a low applied potential of −0.034 V, NADH could be linearly detected from 1.0 mol L−1 up to 6.0 mmol L−1 with a low detection limit of 0.4 mol L−1 (S/N = 3) and fast response time of 2 s. So the PTBO-OMC-IL gel modified electrode may be used as electrochemical transducers and has potential application for designing a variety of NAD+ -dependent electrochemical biosensors. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Ordered mesoporous carbon (OMC) has become a hot subject since it was synthesized in 1999 [1]. Compared with the different forms of carbon materials, OMC possesses special properties (wellordered pore structure, high specific pore volume and high specific surface area) which make it attractive in the field of electroanalytical applications for the low potential determination of different bioanalytes [2,3]. The oxidation of ascorbic acid and uric acid was investigated by Ndamanisha et al. at the OMC functionalized with ferrocenecarboxylic acid modified electrode [4,5]. Recently, studies have been developed by preparing composite films composed of both OMC and conjugated polymers [6,7]. These new composite materials possess the properties of each component with a synergistic effect that would be useful for the detection of electroactive species, including -nicotinamide adenine dinucleotide (NADH) [8], hydrogen peroxide [9] and etc. On the other hand, ionic liquids (IL) have been the targets of numerous investigations because of their characteristics such as good chemical and thermal stability, almost negligible volatility, good ionic conductivity and wide electrochemical window. Hence
∗ Corresponding author. Tel.: +86 537 3196098. E-mail address: [email protected] (X. Zhai). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.12.060
they have been extensively used to prepare modified electrode and biosensors in electrochemistry and biocatalysis. For example, several groups have reported that IL can form gels with carbon nanotubes or OMC by grinding to fabricate several modified electrodes in order to selective detection of dopamine in the presence of ascorbic acid and uric acid [10,11]. NADH and its oxidized form (NAD+ ) are cofactors of more than 500 kinds of dehydrogenase enzymes widely used for the construction of electrochemical biosensors. However, the direct oxidation of NADH at conventional bare electrodes, such as carbon, gold and platinum, is highly irreversible and needs a considerable overpotential [12,13]. Consequently, considerable efforts have been devoted toward the goal of identifying new electrode materials and new methods that will reduce the overpotential for oxidation of NADH and minimize surface passivation effects. Some nanomaterials such as carbon nanotubes (CNT) [14] and OMC [15], water soluble dye compounds [16], phenothiazine derivatives [17] and various redox polymers [18] as electron transfer mediators have been used successfully to decrease the high overpotential for electrocatalytic oxidation of NADH and minimizing surface fouling. Zeng et al. [14] electrodeposited toluidine blue O (TBO) functionalized CNT for a stable low-potential amperometric detection of NADH. Lu et al. [15] fabricated composite film which contains OMC along with the incorporation of poly (neutral red) for determination of NADH.
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In this article, a new gel was developed based on the polymerization of a composite containing TBO functionalized OMC by a – stacking interaction and IL and was used to modify glassy carbon (GC) electrode. The PTBO-OMC-IL gel/GC electrode exhibited high conductivity and good electrocatalytic activity toward NADH due to a positively synergistic effect among PTBO, OMC and IL. 2. Experimental 2.1. Apparatus and reagents All electrochemical experiments were performed with a CHI 660C electrochemical workstation. A conventional three-electrode system was used with a modified grass carbon (GC, 3 mm in diameter) electrode as working electrode, a Ag/AgCl/3 mol L−1 KCl electrode as reference electrode, and a platinum foil electrode as counter electrode. Amperometric measurements were carried out under stirred conditions, and the response current was marked with the change value between the steady state current and the background current. The scanning electron microscopy (SEM) images were performed on a JSM-6360LV SEM (JEOL, Japan). All experiments were performed at room temperature. OMC was synthesized following a published procedure [19]. N,N -dimethylformamide (DMF) (HPLC grade) was purchased from Sangon Biotech Co., Ltd. (China). IL of 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 ) was purchased from Lan zhou Institute of Chemical Physics (China). NADH (reduced form, in the form of sodium salt, >92% purity) was purchased from J&K Scientific Ltd. (China). TBO was purchased from Sigma–Aldrich (USA). McIlvine buffer solution (pH 4.0, consisted of 0.1 mol L−1 citric acid and 0.2 mol L−1 solution of Na2 HPO4 containing 0.1 mol L−1 KCl) was used for electropolymerization and phosphate buffer solution (PBS, 0.067 mol L−1 , pH 7.0) was used as buffer solution for other electrochemical experiments. All other chemicals reagents were of analytical reagent grade and used as received. Double-distilled water was used throughout. 2.2. Preparation of the OMC functionalized with TBO composite The OMC functionalized with TBO (TBO-OMC) composite was firstly prepared by sonicating a mixture of 25 mg OMC and 40 mg TBO in 100 mL DMF for 6 h at room temperature. The resulting suspension was centrifuged with high speed, and then evaporated on a rotary evaporator. Finally, the obtained sample was dried at 75 ◦ C for 5 h to obtain TBO-OMC composite. 2.3. Preparation of the modified electrode Firstly, 12 mg TBO-OMC composite was mixed with 0.16 mL IL by grinding in an agate mortar for about 20 min to form a black TBOOMC-IL gel [20]. Meanwhile, the GC electrode was polished with 0.3 and 0.05 m alumina powder, respectively, followed with being ultrasonically cleaned with ethanol and double-distilled water and dried in nitrogen. Then, the GC electrode was rubbed over the TBOOMC-IL gel for about 15 min placed on a smooth glass slide, and the gel was mechanically attached to the electrode surface. After the gel on the electrode surface was smoothed with a spatula, a thin gel film was left on the GC electrode surface. Finally, the TBOOMC-IL gel modified GC electrode was transferred to a McIlvine buffer solution (pH 4.0) and electropolymerized by cyclic voltammetry between −0.6 and +1.0 V at 50 mV/s for 30 cycles. The begin potential was −0.6 V and the forward scan direction is positive. The electrodeposited gel modified GC electrode (denominated as PTBOOMC-IL gel/GC electrode in this paper) was fabricated. Similarly, the OMC-IL gel could also been formed by grinding 12 mg OMC and
0.16 mL IL in an agate mortar together. Then the OMC-IL gel modified GC electrode was fabricated by the rubbing method, and was denoted as OMC-IL gel/GC electrode hereafter. 3. Results and discussion 3.1. The micrograph of the composite film Fig. 1 displays the morphologies of the OMC, TBO-OMC and PTBO-OMC-IL composite films characterized with SEM. It is clear that OMC dispersed in DMF by sonication is highly entangled. The TBO-OMC composite was also dispersed in DMF by sonication and its image of SEM indicates that the OMC is untangled after functionalized with the TBO as comparison with Fig. 1(A), because TBO is hydrophilic and hydrophobic OMC could interact strongly with TBO by a – stacking interaction. From Fig. 1(C), it can be observed that the PTBO-OMC-IL composite film distributes almost homogeneously with unique structure on the surface of substrate. 3.2. Electropolymerization of TBO and electrochemical characterization of the PTBO-OMC-IL gel/GC electrode According to the reports, the initial step for the TBO polymerization is one-electron oxidation of NH2 group and forms a cation radical [14,21]. The unpaired electron can be delocalization through the TBO molecule, but the unpaired electron may be sited with high probability on either amine group and at position ortho to them. Radical dimerization can occur via carbon–nitrogen coupling routes as shown in Scheme 1. The oxidation of the NH2 group in the dimer can occur again, the polymerization can take place readily to form the PTBO. The consecutive cyclic voltammograms indicate the formation process of the PTBO film on the modified electrode [14]. Fig. 2 shows cyclic voltammograms of the PTBO-OMC-IL gel/GC electrode obtained from the electropolymerization process of TBO with consecutive potential scan. As we can see, the first cyclic voltammogram (initial potential is −0.6 V) exhibits a pair of sharp reversible peaks at the region of the monomer redox peak. The current of the monomer of TBO at about −0.14 V decreased while the current of PTBO at about +0.04 V increased step by step, which means that TBO could be electropolymerized and formed conducting polymer successfully. With increasing scan cycles, a pair of new reversible peaks with a cathodic peak potential of −0.02 V and an anodic peak potential of +0.04 V appear and increase gradually. The above result indicates that the polymer film grows with elapse of time. The cyclic voltammograms responses of the bare GC (curve 1), OMC-IL gel/GC (curve 2) and PTBO-OMC-IL gel/GC (curve 3) electrodes in PBS (0.067 mol L−1 , pH 7.0) are shown in Fig. 3. At the bare GC (curve 1) electrode, no redox peaks could be observed. At the OMC-IL gel/GC electrode (curves 2), a small pair of perks appears which could be ascribed to the redox process of the acidic groups at the surface of OMC [22]. However, the PTBO-OMC-IL gel/GC (curve 3) electrode shows evidently reversible redox responses of the PTBO and the formal potential E1/2 is calculated around at −89.9 mV which is taken as the mid-point of the anodic and cathodic peak potentials. Compared with the other two, the PTBOOMC-IL gel/GC electrode displays the highest background current. To explain the above results, the following three reasons are formulated. Firstly, OMC has larger hydrophobic surface area and could interact strongly with TBO, which is contributed to larger adsorption amount of TBO. Secondly, the OMC-induced enhancement in the behavior of PTBO could be ascribed to improved electronic and ionic transport capacity of the PTBO-OMC-IL gel. Thirdly, PTBO, OMC and IL exhibit some unique characteristics such as high conductivity and good electronic structure, respectively. So, there is a positive synergistic conductibility among PTBO, OMC and IL.
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Fig. 1. SEM images of the OMC (A), TBO-OMC (B) and PTBO-OMC-IL (C) composite flim.
Fig. 4 shows the cyclic voltammograms of the PTBO-OMCIL gel/GC electrode at various scan rates obtained in PBS (0.067 mol L−1 , pH 7.0). It is found that the values of anodic and cathodic peak potentials shift respectively slight to positive and negative directions, and the peak potential separation Ep becomes larger with the increase of scan rate. The Ep is 110 mV at the scan rate of 30 mV s−1 . However, the value changes to 256 mV at the scan rate of 210 mV s−1 . These results demonstrate that the redox peak potentials of the PTBO-OMC-IL gel/GC electrode depend on scan rates. However, the formal potential E1/2 is almost
independent on the scan rates. Fig. 4 inset exhibits that the anodic and cathodic peak currents are both linearly proportional to the scan rates up to 210 mV s−1 , confirming a typical surface-controlled process. When changing the ratio of IL to TBO-OMC with less IL, TBOOMC-IL gel becomes too dense to be easily rubbed over. While changing the ratio of IL to TBO-OMC with more IL, the TBO-OMC-IL gel becomes more diluted in PBS (0.067 mol L−1 , pH 7.0). Besides, the thickness of the modified layer has great impact on the electrochemical properties of the PTBO-OMC-IL/GC electrode. The cyclic
Fig. 2. Cyclic voltammograms of the PTBO-OMC-IL gel/GC electrode obtained from the electropolymerization process of TBO for 30 cycles in McIlvine buffer solution (pH 4.0), scan rate: 50 mV s−1 .
Fig. 3. Cyclic voltammograms of the bare GC (curve 1), OMC-IL gel/GC (curve 2) and PTBO-OMC-IL gel/GC (curve 3) electrodes in PBS (0.067 mol L−1 , pH 7.0), scan rate: 50 mV s−1 .
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Scheme 1. Radical polymerization of TBO.
voltammograms of [Fe(CN)6 ]3−/4− at the modified electrode with different rubbing time are shown in Fig. 5. The charging currents are much larger, as the modified gel gets thicker. When the rubbing time shifts from 0 to 15 min, thickness of the modified layer increases. The currents were found to be almost constant at 15 min rubbing time. Therefore, the optimum rubbing time was chosen as 15 min. The stability of the PTBO-OMC-IL gel/GC electrode was tested by measuring the decrease in voltammetric currents during potential cyclings. Fig. 6 shows almost no decrease in the voltammetric current after continuously scanned for 50 cycles from −0.60 to +0.60 V at a scan rate of 50 mV s−1 in PBS (0.067 mol L−1 , pH 7.0). The good stability of the PTBO-OMC-IL gel/GC electrode may be due to the following reasons. Firstly, the NH2 groups of PTBO and carbon atoms of OMC exhibited a – stacking interaction. Secondly, the gel which contains PTBO functionalized OMC and IL
Fig. 4. Cyclic voltammograms of the PTBO-OMC-IL gel/GC electrode in PBS (0.067 mol L−1 , pH 7.0) at various scan rates: 30, 60, 90, 120, 150, 180 and 210 mV s−1 (from inner to outer). Inset: the relationship between cathodic and anodic peak currents with scan rate.
Fig. 5. Cyclic voltammograms of 5 mM [Fe(CN)6 ]3−/4− containing 0.1 M KCl solution at PTBO-OMC-IL/GC electrode with the rubbing time (1) 0 min, (2) 5 min, (3) 10 min, (4) 15 min and (5) 20 min. Scan rate: 50 mV s−1 .
Fig. 6. Cyclic voltammograms recorded at the PTBO-OMC-IL gel/GC electrode for 50 cycles in PBS (0.067 mol L−1 , pH 7.0), scan rate: 50 mV s−1 .
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Fig. 8. Steady amperometric response of the successive addition of 0.2 mmol L−1 NADH at the bare GC (1), OMC-IL gel/GC (2) and PTBO-OMC-IL gel/GC (3) electrodes in PBS (0.067 mol L−1 , pH 7.0) at the applied potential of −0.034 V.
Fig. 7. Cyclic voltammograms for the bare GC (A), OMC-IL gel/GC (B) and PTBO-OMCIL gel/GC (C) electrodes in PBS (0.067 mol L−1 , pH 7.0), scan rate: 50 mV s−1 . Curves 1 and 2 correspond to the presence and absence of 1.0 mM NADH, respectively, in all cases.
could steadily immobilize matrix on the surface of the modified electrode. 3.3. Electrochemical oxidation of NADH at the PTBO-OMC-IL gel/GC electrode The cyclic voltammograms and amperometric curves of various electrodes can show the electrooxidation of NADH. Fig. 7 compares the cyclic voltammograms for 1.0 mmol L−1 NADH at the bare GC (A), OMC-IL gel/GC (B) and PTBO-OMC-IL gel/GC (C) electrodes in PBS (0.067 mol L−1 , pH 7.0). At the bare GC electrode (Fig. 7A), the oxidation of NADH results in the highest anodic peak potential of about +576 mV. While the anodic peak potential at OMC-IL gel/GC electrode shifts to about +43 mV (Fig. 7B), which is decreased by about 533 mV compared with that of the bare GC electrode. The anodic peak potential at the PTBO-OMCIL gel/GC electrode is apparent at about −36 mV (Fig. 7C), which is reduced by about 612 mV compared with that of the bare GC electrode. Furthermore, the response current is greatly increased at the PTBO-OMC-IL gel/GC electrode compared with the bare GC and OMC-IL gel/GC electrodes, which means the oxidation ability was further increased. Fig. 8 shows the amperometric curves for
successive addition of 0.2 mmol L−1 NADH in a stirred PBS (0.067 mol L−1 , pH 7.0) at the different electrodes. After each addition of NADH, apparent current responses are achieved at all electrodes. The response current of the OMC-IL gel/GC electrode to the addition of 0.2 mmol L−1 NADH is 2.860 A which is 572.0 times as large as that (5.0 nA) obtained at the bare GC electrode. This indicates the OMC-IL gel can improve the current response for electrooxidation of NADH. Especially, the PTBO-OMC-IL gel/GC electrode responds more sensitively and faster to the oxidation of NADH. The response current of the PTBO-OMC-IL gel/GC electrode (Curve 3) to the addition of 0.2 mmol L−1 NADH reaches 3.781 A, which is 1.322 times as large as that obtained at the OMC-IL gel/GC electrode. While the PTBO-OMC-IL gel/GC electrode reduces the response time from 15 s to 2 s for the OMC-IL gel/GC electrode. The analytical parameters for detection of NADH on the corresponding electrodes at the applied potential of −0.034 V are summarized in Table 1. So, the gel integrated with PTBO, OMC and IL influenced the electrode’s voltammetric and amperometric characteristics. The above effects may be explained by the following hypothesis. Firstly, the gel that forms ion-conducting matrix can improve the electronic and ionic transport capacity of PTBO-OMC-IL gel/GC electrode due to the electrooxidation of NADH at lower potential. Secondly, the NADH used in this work is a reduced disodium salt and usually considered as a negative charged molecule in the aqueous solutions. Therefore the charge–charge interaction between the positive charged imidazolium ion (EMIM) and PTBO and the negative charged NADH may be favorable for the electrochemical oxidation of NADH, which resulted in the promotion of the voltammetric and amperometric response. Thirdly, PTBO and OMC have demonstrated outstanding electrocatalytic oxidation toward NADH, respectively [8,14]. So, PTBO, OMC and IL may provide the positively synergistic effect toward the oxidation of NADH. The PTBO-OMC-IL gel/GC electrode was tested at the low applied potential of −0.034 V and it showed good sensitivity toward NADH (12.9 A/mol L−1 ) with good linearity from 1.0 mol L−1 up to 6.0 mmol L−1 (Fig. 9). The detection limit (LOD), based on a signalto-noise ratio of 3, was estimated to be 0.4 mol L−1 . The results compared with the different matrix and mediators recently used for electrocatalytic oxidation of NADH are summarized in Table 2. It suggests that PTBO-OMC-IL gel/GC electrode can be useful for detection of NADH. The stability of the PTBO-OMC-IL gel/GC electrode to the electrocatalytic oxidation of NADH was studied using steady current response measurements method. Fig. 10 displays the amperometric response to 0.2 mmol L−1 NADH, at the applied potential of −0.034 V over a continuous 30-min period. As can be seen, the current diminutions of the bare GC, OMC-IL gel/GC and PTBO-OMC-IL
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Table 1 Analytical parameters at the bare GC, OMC-IL gel/GC and PTBO-OMC-IL gel/GC electrodes for detection of NADH. Electrode
Linear range (mol L−1 )
LOD (mol L−1 )
Sensitivity (A/mmol L−1 )
Response time (s)
Bare GC OMC-IL gel/GC PTBO-OMC- IL gel/GC
50–300 5.0–100 1.0–600
10 2.0 0.4
0.91 8.43 12.9
15 15 2
Table 2 Comparison of the efficiency of some modified electrodes used in the electrocatalysis of NADH. Electrode
Eapp (V)
LOD (M)
Linear range (up to M)
Sensitivity (A mM−1 )
Reference
Poly-BPhM/GC CNT-Chitosan/GC Nile blue/OMC/GC FcC6 SH/MWNTs/GC PTBO-CNT/GC PTBO-OMC-IL gel/GC
+0.2 +0.4 −0.1 +0.65 0.0 −0.034
2 3 1.2 0.54 0.5 0.4
100 300 350 1500 450 600
1.82 – 0.648 – – 12.9
[23] [24] [25] [26] [14] This work
Eapp : applied potential in amperometry.
gel/GC electrode shows good analytical performance such as low potential, fast response time, wide linear range, low detection limit and high sensitivity for determination of NADH, which may have a potential application used as new electrochemical transducer for designing NAD+ -dependent electrochemical biosensors.
Acknowledgments
Fig. 9. Calibration curve between the response current and concentration of NADH at the applied potential of −0.034 V.
The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grant No. 21101075), the Nature Science Foundation of Shandong Province (Grant No. ZR2011BL012 and No. ZR2012BL01), Shandong Province Higher Educational Science and Technology Program (J12LD54), and the Ningxia Medical University Special Talents’ Scientific Research Project (No.XT2012014).
References
Fig. 10. Stability of the current response to 0.2 mmol L−1 NADH at the bare GC (1), OMC-IL gel/GC (2) and PTBO-OMC-IL gel/GC (3) electrodes in PBS (0.067 mol L−1 , pH 7.0) at the applied potential of −0.034 V.
gel/GC electrodes are lower than 2%, 5% and 6%, respectively. The reproducibility of the current response of the PTBO-OMC-IL gel/GC electrode was investigated for 0.2 mmol L−1 NADH and the relative standard deviations was 4.8% (n = 5). 4. Conclusions A novel gel based on electrodeposition of TBO functionalized OMC and IL was successfully prepared and used to modify GC electrode. The PTBO-OMC-IL composite can form a relative uniform film with unique structure on the surface of substrate. This modified electrode displays high conductivity and illustrates the synergic effect on the electrocatalytic oxidation of NADH. The PTBO-OMC-IL
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Biographies Xiurong Zhai received her M.S. degree in Hunan University in 2006. She is currently a lecturer at Department of Chemistry and Chemical Engineering, Jining University. Her current research focuses on electrochemical sensor and biosensor. Yang Cao received his M.Eng. degree in Qingdao Technological University in 2012. He is currently an assistant engineer at Center of Total Pollutants Control, Jining Environmental Protection Bureau, Jining, China. His current research focuses on wetland water purification for engineering research. Guangjun Liu received his Bachelor from Qufu Normal University in 1984. He is currently a professor at Department of Chemistry and Chemical Engineering, Jining University. His current research focuses on electrochemical sensor and biosensor. Yonghong Li received her Ph.D. degree of natural science in Hunan University in 2012. She is currently a lecturer at School of Public Health, Ningxia Medical University. Her current research focuses on electrochemical sensor and biosensor. Hongtao Gao is currently a professor at Chemistry and Chemical Engineering, Jining University. His current research interests are photoelectrocatalysis. Chengyang Yue received her Ph.D. degree of natural science in Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences in 2008. She is an associate professor at Department of Chemistry and Chemical Engineering, Jining University. Her current research focuses on functional materials. Sheng Ning is an associate professor at Department of Chemistry and Chemical Engineering, Jining University. Her current research focuses on compound materials.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electropolymerized toluidine blue O functionalized ordered mesoporous carbon-ionic liquid gel-modified electrode and its low-potential detection of NADH Xiurong Zhai a,∗ , Yonghong Li b , Guangjun Liu a , Yang Cao c , Hongtao Gao a , Chengyang Yue a , Ning Sheng a a
Department of Chemistry and Chemical Engineering, Jining University, Qufu 273155, PR China School of Public Health, Ningxia Medical University, Ningxia, Yinchuan 750004, PR China c Center of Total Pollutants Control, Jining Environmental Protection Bureau, Jining 272000, PR China b
a r t i c l e
i n f o
Article history: Received 9 July 2012 Received in revised form 12 December 2012 Accepted 17 December 2012 Available online 26 December 2012 Keywords: Toluidine blue O Ordered mesoporous carbon Ionic liquid Synergistic effect NADH
a b s t r a c t A novel gel was developed based on the polymerization of a composite containing toluidine blue O (TBO) functionalized ordered mesoporous carbon (OMC) by a – stacking interaction and ionic liquid (IL). The poly (toluidine blue O) (PTBO)-OMC-IL composite film distributes almost homogeneously with unique structure on the surface of substrate. The PTBO-OMC-IL gel modified glass carbon (GC) electrode displays high conductivity investigated by electrochemical method. Electrochemical studies suggest that the PTBO-OMC-IL gel/GC electrode provide a positively synergistic effect among PTBO, OMC and IL on the electrochemical oxidation of NADH. Under a low applied potential of −0.034 V, NADH could be linearly detected from 1.0 mol L−1 up to 6.0 mmol L−1 with a low detection limit of 0.4 mol L−1 (S/N = 3) and fast response time of 2 s. So the PTBO-OMC-IL gel modified electrode may be used as electrochemical transducers and has potential application for designing a variety of NAD+ -dependent electrochemical biosensors. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Ordered mesoporous carbon (OMC) has become a hot subject since it was synthesized in 1999 [1]. Compared with the different forms of carbon materials, OMC possesses special properties (wellordered pore structure, high specific pore volume and high specific surface area) which make it attractive in the field of electroanalytical applications for the low potential determination of different bioanalytes [2,3]. The oxidation of ascorbic acid and uric acid was investigated by Ndamanisha et al. at the OMC functionalized with ferrocenecarboxylic acid modified electrode [4,5]. Recently, studies have been developed by preparing composite films composed of both OMC and conjugated polymers [6,7]. These new composite materials possess the properties of each component with a synergistic effect that would be useful for the detection of electroactive species, including -nicotinamide adenine dinucleotide (NADH) [8], hydrogen peroxide [9] and etc. On the other hand, ionic liquids (IL) have been the targets of numerous investigations because of their characteristics such as good chemical and thermal stability, almost negligible volatility, good ionic conductivity and wide electrochemical window. Hence
∗ Corresponding author. Tel.: +86 537 3196098. E-mail address: [email protected] (X. Zhai). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2012.12.060
they have been extensively used to prepare modified electrode and biosensors in electrochemistry and biocatalysis. For example, several groups have reported that IL can form gels with carbon nanotubes or OMC by grinding to fabricate several modified electrodes in order to selective detection of dopamine in the presence of ascorbic acid and uric acid [10,11]. NADH and its oxidized form (NAD+ ) are cofactors of more than 500 kinds of dehydrogenase enzymes widely used for the construction of electrochemical biosensors. However, the direct oxidation of NADH at conventional bare electrodes, such as carbon, gold and platinum, is highly irreversible and needs a considerable overpotential [12,13]. Consequently, considerable efforts have been devoted toward the goal of identifying new electrode materials and new methods that will reduce the overpotential for oxidation of NADH and minimize surface passivation effects. Some nanomaterials such as carbon nanotubes (CNT) [14] and OMC [15], water soluble dye compounds [16], phenothiazine derivatives [17] and various redox polymers [18] as electron transfer mediators have been used successfully to decrease the high overpotential for electrocatalytic oxidation of NADH and minimizing surface fouling. Zeng et al. [14] electrodeposited toluidine blue O (TBO) functionalized CNT for a stable low-potential amperometric detection of NADH. Lu et al. [15] fabricated composite film which contains OMC along with the incorporation of poly (neutral red) for determination of NADH.
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In this article, a new gel was developed based on the polymerization of a composite containing TBO functionalized OMC by a – stacking interaction and IL and was used to modify glassy carbon (GC) electrode. The PTBO-OMC-IL gel/GC electrode exhibited high conductivity and good electrocatalytic activity toward NADH due to a positively synergistic effect among PTBO, OMC and IL. 2. Experimental 2.1. Apparatus and reagents All electrochemical experiments were performed with a CHI 660C electrochemical workstation. A conventional three-electrode system was used with a modified grass carbon (GC, 3 mm in diameter) electrode as working electrode, a Ag/AgCl/3 mol L−1 KCl electrode as reference electrode, and a platinum foil electrode as counter electrode. Amperometric measurements were carried out under stirred conditions, and the response current was marked with the change value between the steady state current and the background current. The scanning electron microscopy (SEM) images were performed on a JSM-6360LV SEM (JEOL, Japan). All experiments were performed at room temperature. OMC was synthesized following a published procedure [19]. N,N -dimethylformamide (DMF) (HPLC grade) was purchased from Sangon Biotech Co., Ltd. (China). IL of 1-ethyl-3-methylimidazolium tetrafluoroborate (EMIMBF4 ) was purchased from Lan zhou Institute of Chemical Physics (China). NADH (reduced form, in the form of sodium salt, >92% purity) was purchased from J&K Scientific Ltd. (China). TBO was purchased from Sigma–Aldrich (USA). McIlvine buffer solution (pH 4.0, consisted of 0.1 mol L−1 citric acid and 0.2 mol L−1 solution of Na2 HPO4 containing 0.1 mol L−1 KCl) was used for electropolymerization and phosphate buffer solution (PBS, 0.067 mol L−1 , pH 7.0) was used as buffer solution for other electrochemical experiments. All other chemicals reagents were of analytical reagent grade and used as received. Double-distilled water was used throughout. 2.2. Preparation of the OMC functionalized with TBO composite The OMC functionalized with TBO (TBO-OMC) composite was firstly prepared by sonicating a mixture of 25 mg OMC and 40 mg TBO in 100 mL DMF for 6 h at room temperature. The resulting suspension was centrifuged with high speed, and then evaporated on a rotary evaporator. Finally, the obtained sample was dried at 75 ◦ C for 5 h to obtain TBO-OMC composite. 2.3. Preparation of the modified electrode Firstly, 12 mg TBO-OMC composite was mixed with 0.16 mL IL by grinding in an agate mortar for about 20 min to form a black TBOOMC-IL gel [20]. Meanwhile, the GC electrode was polished with 0.3 and 0.05 m alumina powder, respectively, followed with being ultrasonically cleaned with ethanol and double-distilled water and dried in nitrogen. Then, the GC electrode was rubbed over the TBOOMC-IL gel for about 15 min placed on a smooth glass slide, and the gel was mechanically attached to the electrode surface. After the gel on the electrode surface was smoothed with a spatula, a thin gel film was left on the GC electrode surface. Finally, the TBOOMC-IL gel modified GC electrode was transferred to a McIlvine buffer solution (pH 4.0) and electropolymerized by cyclic voltammetry between −0.6 and +1.0 V at 50 mV/s for 30 cycles. The begin potential was −0.6 V and the forward scan direction is positive. The electrodeposited gel modified GC electrode (denominated as PTBOOMC-IL gel/GC electrode in this paper) was fabricated. Similarly, the OMC-IL gel could also been formed by grinding 12 mg OMC and
0.16 mL IL in an agate mortar together. Then the OMC-IL gel modified GC electrode was fabricated by the rubbing method, and was denoted as OMC-IL gel/GC electrode hereafter. 3. Results and discussion 3.1. The micrograph of the composite film Fig. 1 displays the morphologies of the OMC, TBO-OMC and PTBO-OMC-IL composite films characterized with SEM. It is clear that OMC dispersed in DMF by sonication is highly entangled. The TBO-OMC composite was also dispersed in DMF by sonication and its image of SEM indicates that the OMC is untangled after functionalized with the TBO as comparison with Fig. 1(A), because TBO is hydrophilic and hydrophobic OMC could interact strongly with TBO by a – stacking interaction. From Fig. 1(C), it can be observed that the PTBO-OMC-IL composite film distributes almost homogeneously with unique structure on the surface of substrate. 3.2. Electropolymerization of TBO and electrochemical characterization of the PTBO-OMC-IL gel/GC electrode According to the reports, the initial step for the TBO polymerization is one-electron oxidation of NH2 group and forms a cation radical [14,21]. The unpaired electron can be delocalization through the TBO molecule, but the unpaired electron may be sited with high probability on either amine group and at position ortho to them. Radical dimerization can occur via carbon–nitrogen coupling routes as shown in Scheme 1. The oxidation of the NH2 group in the dimer can occur again, the polymerization can take place readily to form the PTBO. The consecutive cyclic voltammograms indicate the formation process of the PTBO film on the modified electrode [14]. Fig. 2 shows cyclic voltammograms of the PTBO-OMC-IL gel/GC electrode obtained from the electropolymerization process of TBO with consecutive potential scan. As we can see, the first cyclic voltammogram (initial potential is −0.6 V) exhibits a pair of sharp reversible peaks at the region of the monomer redox peak. The current of the monomer of TBO at about −0.14 V decreased while the current of PTBO at about +0.04 V increased step by step, which means that TBO could be electropolymerized and formed conducting polymer successfully. With increasing scan cycles, a pair of new reversible peaks with a cathodic peak potential of −0.02 V and an anodic peak potential of +0.04 V appear and increase gradually. The above result indicates that the polymer film grows with elapse of time. The cyclic voltammograms responses of the bare GC (curve 1), OMC-IL gel/GC (curve 2) and PTBO-OMC-IL gel/GC (curve 3) electrodes in PBS (0.067 mol L−1 , pH 7.0) are shown in Fig. 3. At the bare GC (curve 1) electrode, no redox peaks could be observed. At the OMC-IL gel/GC electrode (curves 2), a small pair of perks appears which could be ascribed to the redox process of the acidic groups at the surface of OMC [22]. However, the PTBO-OMC-IL gel/GC (curve 3) electrode shows evidently reversible redox responses of the PTBO and the formal potential E1/2 is calculated around at −89.9 mV which is taken as the mid-point of the anodic and cathodic peak potentials. Compared with the other two, the PTBOOMC-IL gel/GC electrode displays the highest background current. To explain the above results, the following three reasons are formulated. Firstly, OMC has larger hydrophobic surface area and could interact strongly with TBO, which is contributed to larger adsorption amount of TBO. Secondly, the OMC-induced enhancement in the behavior of PTBO could be ascribed to improved electronic and ionic transport capacity of the PTBO-OMC-IL gel. Thirdly, PTBO, OMC and IL exhibit some unique characteristics such as high conductivity and good electronic structure, respectively. So, there is a positive synergistic conductibility among PTBO, OMC and IL.
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Fig. 1. SEM images of the OMC (A), TBO-OMC (B) and PTBO-OMC-IL (C) composite flim.
Fig. 4 shows the cyclic voltammograms of the PTBO-OMCIL gel/GC electrode at various scan rates obtained in PBS (0.067 mol L−1 , pH 7.0). It is found that the values of anodic and cathodic peak potentials shift respectively slight to positive and negative directions, and the peak potential separation Ep becomes larger with the increase of scan rate. The Ep is 110 mV at the scan rate of 30 mV s−1 . However, the value changes to 256 mV at the scan rate of 210 mV s−1 . These results demonstrate that the redox peak potentials of the PTBO-OMC-IL gel/GC electrode depend on scan rates. However, the formal potential E1/2 is almost
independent on the scan rates. Fig. 4 inset exhibits that the anodic and cathodic peak currents are both linearly proportional to the scan rates up to 210 mV s−1 , confirming a typical surface-controlled process. When changing the ratio of IL to TBO-OMC with less IL, TBOOMC-IL gel becomes too dense to be easily rubbed over. While changing the ratio of IL to TBO-OMC with more IL, the TBO-OMC-IL gel becomes more diluted in PBS (0.067 mol L−1 , pH 7.0). Besides, the thickness of the modified layer has great impact on the electrochemical properties of the PTBO-OMC-IL/GC electrode. The cyclic
Fig. 2. Cyclic voltammograms of the PTBO-OMC-IL gel/GC electrode obtained from the electropolymerization process of TBO for 30 cycles in McIlvine buffer solution (pH 4.0), scan rate: 50 mV s−1 .
Fig. 3. Cyclic voltammograms of the bare GC (curve 1), OMC-IL gel/GC (curve 2) and PTBO-OMC-IL gel/GC (curve 3) electrodes in PBS (0.067 mol L−1 , pH 7.0), scan rate: 50 mV s−1 .
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Scheme 1. Radical polymerization of TBO.
voltammograms of [Fe(CN)6 ]3−/4− at the modified electrode with different rubbing time are shown in Fig. 5. The charging currents are much larger, as the modified gel gets thicker. When the rubbing time shifts from 0 to 15 min, thickness of the modified layer increases. The currents were found to be almost constant at 15 min rubbing time. Therefore, the optimum rubbing time was chosen as 15 min. The stability of the PTBO-OMC-IL gel/GC electrode was tested by measuring the decrease in voltammetric currents during potential cyclings. Fig. 6 shows almost no decrease in the voltammetric current after continuously scanned for 50 cycles from −0.60 to +0.60 V at a scan rate of 50 mV s−1 in PBS (0.067 mol L−1 , pH 7.0). The good stability of the PTBO-OMC-IL gel/GC electrode may be due to the following reasons. Firstly, the NH2 groups of PTBO and carbon atoms of OMC exhibited a – stacking interaction. Secondly, the gel which contains PTBO functionalized OMC and IL
Fig. 4. Cyclic voltammograms of the PTBO-OMC-IL gel/GC electrode in PBS (0.067 mol L−1 , pH 7.0) at various scan rates: 30, 60, 90, 120, 150, 180 and 210 mV s−1 (from inner to outer). Inset: the relationship between cathodic and anodic peak currents with scan rate.
Fig. 5. Cyclic voltammograms of 5 mM [Fe(CN)6 ]3−/4− containing 0.1 M KCl solution at PTBO-OMC-IL/GC electrode with the rubbing time (1) 0 min, (2) 5 min, (3) 10 min, (4) 15 min and (5) 20 min. Scan rate: 50 mV s−1 .
Fig. 6. Cyclic voltammograms recorded at the PTBO-OMC-IL gel/GC electrode for 50 cycles in PBS (0.067 mol L−1 , pH 7.0), scan rate: 50 mV s−1 .
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Fig. 8. Steady amperometric response of the successive addition of 0.2 mmol L−1 NADH at the bare GC (1), OMC-IL gel/GC (2) and PTBO-OMC-IL gel/GC (3) electrodes in PBS (0.067 mol L−1 , pH 7.0) at the applied potential of −0.034 V.
Fig. 7. Cyclic voltammograms for the bare GC (A), OMC-IL gel/GC (B) and PTBO-OMCIL gel/GC (C) electrodes in PBS (0.067 mol L−1 , pH 7.0), scan rate: 50 mV s−1 . Curves 1 and 2 correspond to the presence and absence of 1.0 mM NADH, respectively, in all cases.
could steadily immobilize matrix on the surface of the modified electrode. 3.3. Electrochemical oxidation of NADH at the PTBO-OMC-IL gel/GC electrode The cyclic voltammograms and amperometric curves of various electrodes can show the electrooxidation of NADH. Fig. 7 compares the cyclic voltammograms for 1.0 mmol L−1 NADH at the bare GC (A), OMC-IL gel/GC (B) and PTBO-OMC-IL gel/GC (C) electrodes in PBS (0.067 mol L−1 , pH 7.0). At the bare GC electrode (Fig. 7A), the oxidation of NADH results in the highest anodic peak potential of about +576 mV. While the anodic peak potential at OMC-IL gel/GC electrode shifts to about +43 mV (Fig. 7B), which is decreased by about 533 mV compared with that of the bare GC electrode. The anodic peak potential at the PTBO-OMCIL gel/GC electrode is apparent at about −36 mV (Fig. 7C), which is reduced by about 612 mV compared with that of the bare GC electrode. Furthermore, the response current is greatly increased at the PTBO-OMC-IL gel/GC electrode compared with the bare GC and OMC-IL gel/GC electrodes, which means the oxidation ability was further increased. Fig. 8 shows the amperometric curves for
successive addition of 0.2 mmol L−1 NADH in a stirred PBS (0.067 mol L−1 , pH 7.0) at the different electrodes. After each addition of NADH, apparent current responses are achieved at all electrodes. The response current of the OMC-IL gel/GC electrode to the addition of 0.2 mmol L−1 NADH is 2.860 A which is 572.0 times as large as that (5.0 nA) obtained at the bare GC electrode. This indicates the OMC-IL gel can improve the current response for electrooxidation of NADH. Especially, the PTBO-OMC-IL gel/GC electrode responds more sensitively and faster to the oxidation of NADH. The response current of the PTBO-OMC-IL gel/GC electrode (Curve 3) to the addition of 0.2 mmol L−1 NADH reaches 3.781 A, which is 1.322 times as large as that obtained at the OMC-IL gel/GC electrode. While the PTBO-OMC-IL gel/GC electrode reduces the response time from 15 s to 2 s for the OMC-IL gel/GC electrode. The analytical parameters for detection of NADH on the corresponding electrodes at the applied potential of −0.034 V are summarized in Table 1. So, the gel integrated with PTBO, OMC and IL influenced the electrode’s voltammetric and amperometric characteristics. The above effects may be explained by the following hypothesis. Firstly, the gel that forms ion-conducting matrix can improve the electronic and ionic transport capacity of PTBO-OMC-IL gel/GC electrode due to the electrooxidation of NADH at lower potential. Secondly, the NADH used in this work is a reduced disodium salt and usually considered as a negative charged molecule in the aqueous solutions. Therefore the charge–charge interaction between the positive charged imidazolium ion (EMIM) and PTBO and the negative charged NADH may be favorable for the electrochemical oxidation of NADH, which resulted in the promotion of the voltammetric and amperometric response. Thirdly, PTBO and OMC have demonstrated outstanding electrocatalytic oxidation toward NADH, respectively [8,14]. So, PTBO, OMC and IL may provide the positively synergistic effect toward the oxidation of NADH. The PTBO-OMC-IL gel/GC electrode was tested at the low applied potential of −0.034 V and it showed good sensitivity toward NADH (12.9 A/mol L−1 ) with good linearity from 1.0 mol L−1 up to 6.0 mmol L−1 (Fig. 9). The detection limit (LOD), based on a signalto-noise ratio of 3, was estimated to be 0.4 mol L−1 . The results compared with the different matrix and mediators recently used for electrocatalytic oxidation of NADH are summarized in Table 2. It suggests that PTBO-OMC-IL gel/GC electrode can be useful for detection of NADH. The stability of the PTBO-OMC-IL gel/GC electrode to the electrocatalytic oxidation of NADH was studied using steady current response measurements method. Fig. 10 displays the amperometric response to 0.2 mmol L−1 NADH, at the applied potential of −0.034 V over a continuous 30-min period. As can be seen, the current diminutions of the bare GC, OMC-IL gel/GC and PTBO-OMC-IL
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Table 1 Analytical parameters at the bare GC, OMC-IL gel/GC and PTBO-OMC-IL gel/GC electrodes for detection of NADH. Electrode
Linear range (mol L−1 )
LOD (mol L−1 )
Sensitivity (A/mmol L−1 )
Response time (s)
Bare GC OMC-IL gel/GC PTBO-OMC- IL gel/GC
50–300 5.0–100 1.0–600
10 2.0 0.4
0.91 8.43 12.9
15 15 2
Table 2 Comparison of the efficiency of some modified electrodes used in the electrocatalysis of NADH. Electrode
Eapp (V)
LOD (M)
Linear range (up to M)
Sensitivity (A mM−1 )
Reference
Poly-BPhM/GC CNT-Chitosan/GC Nile blue/OMC/GC FcC6 SH/MWNTs/GC PTBO-CNT/GC PTBO-OMC-IL gel/GC
+0.2 +0.4 −0.1 +0.65 0.0 −0.034
2 3 1.2 0.54 0.5 0.4
100 300 350 1500 450 600
1.82 – 0.648 – – 12.9
[23] [24] [25] [26] [14] This work
Eapp : applied potential in amperometry.
gel/GC electrode shows good analytical performance such as low potential, fast response time, wide linear range, low detection limit and high sensitivity for determination of NADH, which may have a potential application used as new electrochemical transducer for designing NAD+ -dependent electrochemical biosensors.
Acknowledgments
Fig. 9. Calibration curve between the response current and concentration of NADH at the applied potential of −0.034 V.
The authors gratefully acknowledge the financial support of the National Natural Science Foundation of China (Grant No. 21101075), the Nature Science Foundation of Shandong Province (Grant No. ZR2011BL012 and No. ZR2012BL01), Shandong Province Higher Educational Science and Technology Program (J12LD54), and the Ningxia Medical University Special Talents’ Scientific Research Project (No.XT2012014).
References
Fig. 10. Stability of the current response to 0.2 mmol L−1 NADH at the bare GC (1), OMC-IL gel/GC (2) and PTBO-OMC-IL gel/GC (3) electrodes in PBS (0.067 mol L−1 , pH 7.0) at the applied potential of −0.034 V.
gel/GC electrodes are lower than 2%, 5% and 6%, respectively. The reproducibility of the current response of the PTBO-OMC-IL gel/GC electrode was investigated for 0.2 mmol L−1 NADH and the relative standard deviations was 4.8% (n = 5). 4. Conclusions A novel gel based on electrodeposition of TBO functionalized OMC and IL was successfully prepared and used to modify GC electrode. The PTBO-OMC-IL composite can form a relative uniform film with unique structure on the surface of substrate. This modified electrode displays high conductivity and illustrates the synergic effect on the electrocatalytic oxidation of NADH. The PTBO-OMC-IL
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Biographies Xiurong Zhai received her M.S. degree in Hunan University in 2006. She is currently a lecturer at Department of Chemistry and Chemical Engineering, Jining University. Her current research focuses on electrochemical sensor and biosensor. Yang Cao received his M.Eng. degree in Qingdao Technological University in 2012. He is currently an assistant engineer at Center of Total Pollutants Control, Jining Environmental Protection Bureau, Jining, China. His current research focuses on wetland water purification for engineering research. Guangjun Liu received his Bachelor from Qufu Normal University in 1984. He is currently a professor at Department of Chemistry and Chemical Engineering, Jining University. His current research focuses on electrochemical sensor and biosensor. Yonghong Li received her Ph.D. degree of natural science in Hunan University in 2012. She is currently a lecturer at School of Public Health, Ningxia Medical University. Her current research focuses on electrochemical sensor and biosensor. Hongtao Gao is currently a professor at Chemistry and Chemical Engineering, Jining University. His current research interests are photoelectrocatalysis. Chengyang Yue received her Ph.D. degree of natural science in Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences in 2008. She is an associate professor at Department of Chemistry and Chemical Engineering, Jining University. Her current research focuses on functional materials. Sheng Ning is an associate professor at Department of Chemistry and Chemical Engineering, Jining University. Her current research focuses on compound materials.