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Hollow nitrogen-doped carbon microspheres pyrolyzed from self-polymerized dopamine and its application in simultaneous electrochemical determination of uric acid, ascorbic acid and dopamine
Biosensors and Bioelectronics 26 (2011) 2934–2939
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Hollow nitrogen-doped carbon microspheres pyrolyzed from self-polymerized dopamine and its application in simultaneous electrochemical determination of uric acid, ascorbic acid and dopamine Chunhui Xiao, Xiaochen Chu, Yan Yang, Xing Li, Xiaohua Zhang, Jinhua Chen ∗ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Lushan Southern Road, Changsha 410082, PR China
a r t i c l e
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Article history: Received 23 August 2010 Received in revised form 26 November 2010 Accepted 26 November 2010 Available online 4 December 2010 Keywords: Hollow nitrogen-doped carbon microspheres Dopamine Ascorbic acid Uric acid Differential pulse voltammetry
a b s t r a c t Hollow nitrogen-doped carbon microspheres (HNCMS) as a novel carbon material have been prepared and the catalytic activities of HNCMS-modified glassy carbon (GC) electrode towards the electrooxidation of uric acid (UA), ascorbic acid (AA) and dopamine (DA) have also been investigated. Comparing with the bare GC and carbon nanotubes (CNTs) modified GC (CNTs/GC) electrodes, the HNCMS modified GC (HNCMS/GC) electrode has higher catalytic activities towards the oxidation of UA, AA and DA. Moreover, the peak separations between AA and DA, and DA and UA at the HNCMS/GC electrode are up to 212 and 136 mV, respectively, which are superior to those at the CNTs/GC electrode (168 and 114 mV). Thus the simultaneous determination of UA, AA and DA was carried out successfully. In the co-existence system of UA, AA and DA, the linear response range for UA, AA and DA are 5–30 M, 100–1000 M and 3–75 M, respectively and the detection limits (S/N = 3) are 0.04 M, 0.91 M and 0.02 M, respectively. Meanwhile, the HNCMS/GC electrode can be applied to measure uric acid in human urine, and may be useful for measuring abnormally high concentration of AA or DA. The attractive features of HNCMS provide potential applications in the simultaneous determination of UA, AA and DA. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Simultaneous determination of uric acid (UA), dopamine (DA) and ascorbic acid (AA) has received a great deal of attention (Xiao et al., 2007). Since UA, AA and DA are highly electrochemically active, electrochemical methods for their simultaneous determination have the inherent advantages of simplicity, rapidity, and high sensitivity. However, UA, AA and DA are oxidized at similar potentials with poor sensitivity at bare solid electrodes and the overlap of their voltammetric responses would confuse their simultaneous determination. In addition, the products of oxidation can be absorbed or electropolymerized onto the electrode surface, promoting its partial passivation and negatively affecting detectability and reusability of electrodes (Chen et al., 2009; Gonon et al., 1980; Lane and Blaha, 1990). To overcome these problems, various modified electrodes have been developed, such as polymer film modified electrodes (Cizewski and Milczarek, 1999; Gao and Huang, 1998; Kalimuthu and John, 2010; Lin et al., 2008a), nanoparticles modified electrodes (Huang et al., 2008; Shakkthivel and Chen, 2007; Tang et al., 2008;
∗ Corresponding author. Tel.: +86 731 88821818; fax: +86 731 88821818. E-mail addresses: [email protected], [email protected] (J. Chen). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.11.041
Weng et al., 2005), electrochemically oxidized glassy carbon (GC) electrode (Thiagarajan et al., 2009), pyrolytic graphite electrode (Silva et al., 2008), screen-printed carbon electrode (Prasad et al., 2008), carbon ionic liquid electrode (Safavi et al., 2006), carbon nanohorn modified electrode (Zhu et al., 2009) and carbon nanotube modified electrodes (Deng et al., 2009; Hocevar et al., 2005; Kumar et al., 2010; Liu et al., 2007; Poh et al., 2004; Wang et al., 2002). Among these electrodes, carbon material based electrode is considered as one of the most effective electrodes to carry out the simultaneous determination of UA, AA and DA due to their remarkable electrocatalytic properties. On the other hand, hollow carbon microspheres (HCMS) with a structure of hollow cores and carbon shells have high specific surface area, low density, high surface permeability and good electronic properties, currently attract intense interest in drug delivery, active material encapsulation, lithium-ion battery, catalyst supports, hydrogen storage, damping materials, and templates for the synthesis of other useful hollow materials (Du et al., 2007; Gill and Ballesteros, 1998; Han et al., 2003; Huang et al., 1999; Su et al., 2006; Sun and Li, 2004; Wen et al., 2007). However, there are few works focused on their application in electrochemical biosensors. The synthesis methods used to produce HCMS include chemical vapor deposition (CVD), the catalytic-reduction route, the sol–gel method, the solid template approach and the solvothermal method
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(Dai et al., 2000; Kizukaa et al., 2009; Li et al., 2008; Ni et al., 2005; Xia and Mokaya, 2004; Xia et al., 2004; Xiong et al., 2003). It is noted that these methods are commonly carried out with multiple procedures or specific equipments. Other cases, such as the pyrolysis of the mixed carbon sources, usually led to the production of smaller proportion or less homogeneous HCMS (Liu et al., 2003). Based on the good features of HCMS and the deficiencies in the existing synthesis methods, we expect to find a simple, low-cost and effective approach to prepare HCMS and explore their application in simultaneous electrochemical determination of UA, AA and DA. It is well known that pyrolysis of core/shell polymer particles is a versatile method to obtain HCMS, whose pore size and shell thickness can be easily tailored by adjusting the core size and shell thickness of the core/shell polymers. Besides, it offers a simple way to introduce heteroatoms into carbon-based materials. Heteroatoms (such as nitrogen), which are present in the carbon materials, change the electron donor/acceptor characteristics of carbon depending on their chemical states, thus leading to an enhancement of electrochemical properties (Kima et al., 2008; Lin et al., 2008b; Shao et al., 2010). Herein, using DA as the source of carbon and nitrogen, we successfully synthesized the hollow nitrogen-doped carbon microspheres (HNCMS) by pyrolysis of polydopamine (PDA) wrapped SiO2 microspheres which were fabricated by the spontaneous selfpolymerization of dopamine onto silica microspheres. This method is versatile, relatively quick, and inexpensive. It was found that the HNCMS modified GC electrode had high electrochemical catalytic activities towards the oxidation of UA, AA and DA due to the hollow structure, good electrochemical properties and nitrogen-doping of the HNCMS. And in the co-existence system of UA, AA and DA, the simultaneous determination of UA, AA and DA could be simply achieved, which contributed greatly to the practical analysis of UA, AA and DA. 2. Experimental 2.1. Reagents Dopamine hydrochloride and SiO2 particles (diameter, ∼360 nm) were purchased from Alfa Aesar (USA) and used as received. Multi-walled carbon nanotubes (CNTs, >95%, diameter 20–40 nm, length 5–15 m) prepared by CVD were purchased from Shenzhen Nanotech Port Ltd. and purified by refluxing in concentrated nitric acid solution for 6 h. All other chemicals were of analytical grade. Aqueous solutions used throughout were prepared with ultra-pure water obtained from a Millipore system (>18 M cm). All electrochemical measurements were carried out at room temperature (25 ± 2 ◦ C). 2.2. Apparatus The scanning electron microscope (SEM) observations were conducted on JSM 6700F (JEOL, Japan). Nitrogen sorption isotherms and Brunauer–Emmett–Teller (BET) surface areas of the materials were determined by an ASAP 2010 Micrometrics sorptometer (America). Nitrogen content in HNCMS was determined by elemental analyzer (TCH-600, America). All electrochemical measurements were performed on a CHI 660D electrochemical workstation (Chenhua Instrument Company of Shanghai, China) with a conventional three-electrode system comprising a platinum wire as an auxiliary electrode, a saturated calomel electrode (SCE) as the reference electrode, and modified GC electrode as the working electrode. All the potentials in this paper were in respect to SCE.
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2.3. Preparation of HNCMS The preparation of HNCMS was composed of three steps and the schematic procedure for the synthesis of the HNCMS is shown in Scheme 1. PDA/SiO2 microspheres were synthesized according to the literature (Postma et al., 2009). In brief, 100 mg silica particles were washed thoroughly with 50 mM tris(hydroxymethyl)aminomethane (TRIS) buffer (pH = 8.5) and centrifuged three times, respectively. The deposit was resuspended in 40 mL 50 mM TRIS buffer (pH = 8.5) contained 2 mg mL−1 DA, followed by vigorous stirring for 24 h to form PDA/SiO2 microspheres (the molecular structure transformation during self-polymerization is shown in Scheme 1a). Then deep brown particles were centrifuged (3000 rpm, 1 min) and washed with fresh TRIS buffer (three times) to remove the tan colored solution, followed by drying in vacuum at 60 ◦ C overnight. The nitrogen-doped carbon/SiO2 microspheres were prepared by pyrolysis of the obtained PDA/SiO2 microspheres in a nitrogen atmosphere at 800 ◦ C for 2 h (Scheme 1b). Finally, the HNCMS were obtained via removal of the template (silica core) in 2 M HF + 8 M NH4 F solution at room temperature for 10 min, followed by successive centrifugation (13,000 rpm, 5 min) and washing three times, and drying in vacuum at 60 ◦ C overnight (Scheme 1c). The nitrogen content in the HNCMS was detected by elemental analyzer and is about 7.28 wt.%. 2.4. Preparation of HNCMS modified electrode Prior to use, GC electrodes (diameter = 3 mm) were carefully polished to a mirror-like plane with 0.5 and 0.05-m alumina slurries, successively. Afterward, the electrode was washed thoroughly with excess amounts of water and dried under nitrogen gas. The HNCMS modified GC (HNCMS/GC) electrode was prepared by casting 5 L HNCMS suspension (1 mg mL−1 HNCMS in N,Ndimethylformamide) on the surface of GC electrode and dried at room temperature for 24 h. For comparison, the CNTs/GC electrode was also prepared under the same procedure. For real sample analysis, no pretreatment process was performed. 25 L of the urine sample was diluted with 10 mL 0.1 M PBS (pH 7.0) and then the original concentration of uric acid in this diluted human urine was detected and is 14.16 M (Table S1, see the supplementary information). 3. Results and discussion 3.1. Characterization of HNCMS The morphology of the as-prepared samples was characterized by SEM. Fig. S1 (see the supplementary information) and Fig. 1 shows the SEM images of the SiO2 microspheres, PDA/SiO2 microspheres, carbon/SiO2 microspheres and HNCMS. From Fig. S1(A), it is noted that the diameter of SiO2 microspheres is about 360 nm. After 24 h self-polymerization of DA, PDA wrapped SiO2 microspheres with diameter of about 400 nm can be observed in Fig. S1(B) and the PDA shell is about 40 nm. Following carbonization in nitrogen atmosphere at 800 ◦ C for 2 h, the nitrogen-doped carbon/SiO2 composite is obtained. From Fig. S1(C), it is noted that the diameter of microsphere is decreased by ca. 5% due to the pyrolysis of PDA. Fig. 1 shows the SEM image of the HNCMS after removal of SiO2 template and the inset is the SEM image at higher magnification. The hollow feature of the microspheres with uniform diameter (∼380 nm) and shell thickness (∼20 nm) can be clearly observed. This indicates that structurally robust and replicated HNCMS can be fabricated successfully according to the procedure shown in Scheme 1.
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Scheme 1. Schematic routine for preparation of the hollow N-doped carbon microspheres.
Representative nitrogen adsorption/desorption isotherms and the corresponding Barrett–Joyner–Halenda (BJH) pore size distribution curve of the obtained HNCMS are shown in Fig. S2 (see the supplementary information). The nitrogen sorption isotherm of the product exhibits typical IV isotherm with H1 hysteresis at high relative pressure, which means that the obtained HNCMS possess mesoporous structure (Vinu et al., 2005). The pore-size distribution of HNCMS was calculated from nitrogen desorption using the BJH model, where the results show a distribution centered at 3.9 nm. The BET surface area of the HNCMS was calculated from the results of nitrogen adsorption and is 639.58 m2 g−1 , which is much higher than that of other HCMS (Wang et al., 2005; Wu et al., 2006) and CNTs (40–300 m2 g−1 ). Based on the hollow structure, high specific
Fig. 1. SEM image of hollow N-doped carbon microspheres. Inset: the magnified image on the corresponding area.
surface area and N-doping, HNCMS may have good electrochemical properties. 3.2. Electrochemical properties of HNCMS Fe(CN)6 3−/4− , as an electrochemical probe, is usually used to evaluate the electrochemical properties of the electrode. Fig. 2 shows cyclic voltammograms obtained at the bare GC, CNTs/GC and HNCMS/GC electrodes in 5 mM Fe(CN)6 3−/4− (1:1) + 0.1 M KCl solution. It is noted that the difference in potential between the anodic and cathodic peaks (Ep ) is 73 mV for the HNCMS/GC electrode, 85 mV for the CNTs/GC electrode and 110 mV for the bare GC electrode. As Ep is the function of the electron transfer rate, the
Fig. 2. CVs obtained at the bare GC (a), CNTs/GC (b) and HNCMS/GC (c) electrodes in 5 mM Fe(CN)6 3−/4− (1:1) + 0.1 M KCl aqueous solution. Scan rate: 50 mV s−1 .
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Fig. 3. (A) CVs at the bare GC (a1), CNTs/GC (b1) and HNCMS/GC electrodes (c1) in 0.1 M PBS containing 200 M UA. (B) CVs at the bare GC (d1), CNTs/GC (e1) and HNCMS/GC electrodes (f1) in 0.1 M PBS containing 1 mM AA. (C) CVs at the bare GC (g1), CNTs/GC (h1) and HNCMS/GC electrodes (i1) in 0.1 M PBS containing 100 M DA. Scan rate: 50 mV s−1 . Inset: the corresponding DPVs in 0.1 M PBS containing UA, AA and DA. (D) DPVs at the bare GC (j), CNTs/GC (k) and HNCMS/GC electrodes (l) in 0.1 M PBS containing 100 M UA + 500 M AA + 100 M DA.
lower Ep , the higher electron transfer rate. The electron transfer rate constant k0 for the ferricyanide system decreases with the increase of the value of Ep (Nicholson, 1965; Jia et al., 2007). Therefore, the order of the k0 value at different electrodes is as follows: HNCMS/GC > CNTs/GC > bare GC. Furthermore, the redox peak currents at the HNCMS/GC electrode are larger than that at the CNTs/GC and bare GC electrodes. This may result from the good electronic conductivity and large specific surface area of HNCMS. The smaller value of Ep and the higher redox peak currents indicate that the HNCMS/GC electrode has better electrochemical properties than the CNTs/GC and bare GC electrodes. The capability of electron transfer of different electrodes was further investigated by electrochemical impedance analysis and the corresponding results are shown in Fig. S3 (see the supplementary information). The order of the value of chargetransfer resistance (Rct ) for different electrodes is as follows: bare GC > CNTs/GC > HNCMS/GC. This result is in accordance with that observed in Fig. 2 and reconfirms that HNCMS possess good conductivity and electrochemical properties. These imply that HNCMS may be a promising electrochemical active material to construct electrochemical biosensors. 3.3. Electrocatalytic oxidation of UA, AA and DA Fig. 3 shows the cyclic voltammograms (CVs) of UA, AA and DA oxidation at the different electrodes and the inset plots show the corresponding differential pulse voltammetry (DPV) responses. For UA (200 M) oxidation, it can be seen from Fig. 3(A) that the oxidation peak potential at all of the electrodes appears at about 335 mV. However, the DPV peak current at the HNCMS/GC electrode (77.0 A) is 3.5 times and 22 times as high as that at the CNTs/GC electrode (22.0 A) and the bare GC electrode (3.5 A), respectively. The reversibility of the electrode process is not good although a weak reduction peak can be observed at the HNCMS/GC
electrode. Fig. 3(B) shows the electro-oxidation of 1 mM AA at the HNCMS/GC, CNTs/GC and bare GC electrodes. It is noted that AA oxidation occurs irreversibly with a large overpotential (326 mV) at the bare GC electrode, indicating the slow electron transfer because of the electrode fouling caused by the adsorption of oxidation products of AA (Zhang et al., 2005). Although both the oxidation potentials of AA at the HNCMS and CNTs modified GC electrodes shift negatively, oxidation potential at the HNCMS/GC electrode (−10 mV) is more negative than that at the CNTs/GC electrode (35 mV). Moreover, the DPV peak current at the HNCMS/GC electrode (39.8 A) shows 2 and 6 times higher than that at the CNTs/GC (19.8 A) and bare GC electrodes (6.6 A), respectively. In the case of AA, no reduction peak is observed at all electrodes, which show irreversible electrode processes for AA electro-oxidation. For 100 M DA (Fig. 3(C)), it can be found that a couple of the weak redox peaks with a Ep of 80 mV are observed at the bare GC electrode. The oxidation peak should result from the oxidation of dopamine to doaminequinone, while the reduction peak is caused by reducing doaminequinone to dopamine. However, a couple of the reversible and well-defined redox peaks with a Ep of 30 mV can be observed at both HNCMS/GC and CNTs/GC electrodes. In addition, the DPV peak current at the HNCMS/GC electrode (81.6 A) is 1.6 and 13.6 times higher than that at the CNTs/GC (49.6 A) and bare GC electrodes (6.0 A), respectively. These results indicate that the HNCMS/GC electrode possesses the best electrocatalytic properties towards the oxidation of UA, AA and DA among these three electrodes, especially negatively shift of the AA oxidation potential. This may be beneficial to simultaneous determination of UA, AA and DA with high sensitivity and excellent selectivity. In order to show clearly the ability of the electrode for the simultaneous determination of UA, AA and DA, the DPV results obtained in the co-existence system of UA, AA and DA are shown in Fig. 3(D). For the bare GC electrode, the oxidation peaks of AA
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and DA completely overlap and show a broad overlapped peak at 260 mV. The oxidation peak of UA appears at 392 mV and partially overlaps with that of AA and DA. At the CNTs/GC electrode, the DPV peak potentials are 296 mV, 14 mV, and 182 mV for UA, AA and DA, respectively. In contrast, the DPV peaks are well-resolved at the HNCMS/GC electrode with the peak potentials at about 292 mV, −56 mV, and 156 mV for UA, AA and DA, respectively. The peak separations for UA–DA and DA–AA at the HNCMS/GC electrode are up to 212 and 136 mV, respectively, which is superior to that at the CNTs/GC electrode (168 and 114 mV). These results reaffirm that HNCMS not only enhance the oxidation of UA, AA and DA, but also dramatically enlarge the peak separation among UA, AA and DA. The enlarged separation of the anodic peak potential, coupled with the increased sensitivity, indicates that the simultaneous determination of UA, AA and DA in the co-existence system is feasible. Also it is believed that the HNCMS modified electrode can be used for some interesting biological applications. 3.4. Determination of UA, AA and DA Since DPV has much higher current sensitivity and better resolution compared to CV, simultaneous determination of UA, AA and DA at HNCMS/GC electrode was carried out using DPV method. In ternary mixture, concentration of one species changed, and those of other two species remained constant. From Fig. 4(A), the peak current of UA in 0.1 M PBS containing 200 M AA and 40 M DA increases linearly with the increase of the UA concentration from 5 to 30 M. The corresponding linear function is Ip,UA (A) = 0.41 + 0.59 CUA (M) with a correlation coefficient of R = 0.9988 (relative standard deviations (R.S.D.) (n = 9) of responses for AA and DA are 1.7% and 3.3%, respectively). Similarly, the oxidation current of AA in 0.1 M PBS containing 80 M UA and 80 M DA increases with the increase of the AA concentration (Fig. 4(B)) and the linear range is 100–1000 M with the linear function Ip,AA (A) = 0.68 + 0.03 CAA (M) (R = 0.9989) (R.S.D. (n = 9) of responses for UA and DA are 2.3% and 2.6%, respectively). From Fig. 4(C), it can also be observed that the peak current of DA in 0.1 M PBS containing 80 M UA and 500 M AA increases linearly with the increase of the DA concentration from 3 to 70 M and the linear function is Ip,DA (A) = 1.14 + 0.93 CDA (M) with correlation of R = 0.9979 (R.S.D. (n = 10) of responses for UA and AA are 3.1% and 2.1%, respectively). The detection limits (S/N = 3) for UA, AA and DA are 0.04 M, 0.91 M and 0.02 M, respectively. Comparing the data shown in the previous literatures (Table 1), improved or comparable performance for the simultaneous determination of UA, AA and DA can be achieved using the HNCMS/GC electrode. Especially for AA, the detection limit by this method is improved mostly by more than one order of magnitude. To our best knowledge, direct electrochemical determination of AA in the co-existence system of UA, AA and DA with such a low detection limit is rarely reported. In order to investigate the stability of the electrode, the DPV responses in ternary mixture solution containing 80 M UA, 500 M AA and 80 M DA were recorded for 6 successive measurements. The current responses for six injections remained almost constant and the relative standard deviations (R.S.D.) of the same electrode were 2.0%, 0.7%, and 2.2% for UA, AA and DA, respectively. Also, there were no obvious decreases (3.1%, 1.8% and 3.7% for 80 M UA, 500 M AA and 80 M DA, respectively) in the responses of the HNCMS/GC electrode after its storage in PBS for a week. All those indicate that the stability of the HNCMS/GC electrode is good. Meanwhile, the analytical application of the HNCMS/GC electrode has been investigated by applying the HNCMS/GC electrode to determine UA, AA and DA in human urine by the calibration curve method, and the results are shown in Table S1 (see the supplementary information). The urine sample was diluted 400 times with 0.1 M PBS before the measurement to fit the calibration curve and
Fig. 4. DPVs at the HNCMS/GC electrode in 0.1 M PBS (A) containing 200 M AA, 40 M DA and different concentrations of UA (from inner to outer): 0, 5, 10, 20, 30, 50, 70, 110, 150 M; (B) containing 80 M UA, 80 M DA and different concentrations of AA (from inner to outer): 0, 100, 200, 300, 400, 500, 600, 800, 1000 M and (C) containing 80 M UA, 500 M AA and different concentrations of DA (from inner to outer): 0, 3, 6, 15, 25, 40, 55, 70, 100 and 150 M. Insets: plots of Ip vs. concentration for UA, AA and DA, respectively.
also reduce the matrix effect. No other pretreatment process was performed. It is noted that the spike recoveries of 105.2%, 97.8% and 99.5% for 10 M UA, 20 M DA and 200 M AA respectively are satisfactory, implying that the proposed electrochemical sensor (the HNCMS/GC electrode) may have great potential application for simultaneous determination of UA, AA and DA in real samples.
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Table 1 Comparison of the analytical performance of the different modified electrodes for the simultaneous determination of AA, DA and UA. Electrode materials
Oracet blue f-MWNT PGE Oxidized GCE Poly-ACBK SWCNH CNT/PEDOT HNCMS a
Linear range (M)
LODa (M)
Sensitivity (A/M)
Reference
UA
AA
DA
UA
AA
DA
UA
AA
DA
20–600 75–180 2.5–30 1.97–9.88 1.0–120.0 0.06–10 10–250 3–30
40–800 75–187 25–500 197–988 50.0–1000 30–400 100–2000 100–1000
0.06–0.8 75–180 1–20 1.97–9.88 1.0–200.0 0.2–3.8 10–330 5–70
0.0071 0.08 1.52 0.0833 0.1311 5.95 – 0.59
0.0022 0.027 0.012 0.00009 0.0137 0.020 – 0.027
0.147 0.146 1.87 3.1575 0.1234 3.49 – 0.93
0.4 – 1.4
1.3 – 13
0.02 – 0.1
0.5 0.02 10 0.04
10.0 5 100 0.91
0.5 0.06 10 0.02
Zare et al. (2006) Yogeswaran and Chen (2007) Silva et al. (2008) Thiagarajan et al. (2009) Zhang et al. (2009) Zhu et al. (2009) Lin et al. (2010) This work
Limit of detection.
4. Conclusions Hollow nitrogen-doped carbon microspheres (HNCMS) were fabricated by pyrolysis of self-polymerized dopamine, and a novel and simple strategy for simultaneous determination of UA, AA and DA based on the HNCMS/GC electrode is presented. It was found that the electrochemical behaviors of UA, AA and DA have been improved at the HNCMS/GC electrode, which is ascribed to the unique hollow structure of HNCMS. Furthermore, compared with the CNTs modified GC electrode, the HNCMS/GC electrode shows more effective simultaneous determination of UA, AA and DA in coexistence system due to the larger separation of the oxidation peak potentials of UA, AA and DA. Additionally, the HNCMS/GC electrode has good stability. This study indicates a great promising of HNCMS in analytical applications. Acknowledgments This work was financially supported by the National Basic Research Program of China (No. 2009CB421601), NSFC (20975033, 20905024) and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2010.11.041. References Chen, P.Y., Vittal, R., Nien, P.C., Ho, K.C., 2009. Biosens. Bioelectron. 24, 3504–3509. Cizewski, A., Milczarek, G., 1999. Anal. Chem. 71, 1055–1061. Dai, K., Toshiaki, A., Yoshio, K., Akira, T., 2000. Chem. Mater. 12, 3397–3401. Deng, C.Y., Chen, J.H., Wang, M.D., Xiao, C.H., Nie, Z., Yao, S.Z., 2009. Biosens. Bioelectron. 24, 2091–2094. Du, H.D., Li, B.H., Kang, F.Y., Fu, R.W., Zeng, Y.Q., 2007. Carbon 45, 429–435. Gao, Z.Q., Huang, H., 1998. Chem. Commun., 2107–2108. Gill, I., Ballesteros, A., 1998. J. Am. Chem. Soc. 128, 8587–8592. Gonon, F., Buda, M., Cespuglio, R., Jouvert, M., Pajol, J., 1980. Nature 286, 902–904. Han, S.G., Yun, Y.K., Park, K.W., Sung, Y.E., Hyeon, T., 2003. Adv. Mater. 15, 1922–1925. Hocevar, S.B., Wang, J., Deo, R.P., Musameh, M., Ogorevc, B., 2005. Electroanalysis 17, 417–422. Huang, H.Y., Remsen, E.E., Kowalewski, T., Wooley, K.L., 1999. J. Am. Chem. Soc. 121, 3805–3806. Huang, J.S., Liu, Y., Hou, H.Q., You, T.Y., 2008. Biosens. Bioelectron. 24, 632–637. Jia, N.Q., Wang, Z.Y., Yang, G.F., Shen, H.B., Zhu, L.Z., 2007. Electrochem. Commun. 9, 233–238.
Kalimuthu, P., John, S.A., 2010. Talanta 80, 1686–1691. Kima, N.D., Kima, W., Joo, J.B., Oha, S., Kimb, P., Kimc, Y., Yi, J., 2008. J. Power Sources 180, 671–675. Kizukaa, T., Katoa, R., Miyazawab, K., 2009. Carbon 47, 138–144. Kumar, S.A., Wang, S.F., Yang, T.C.K., Yeh, C.T., 2010. Biosens. Bioelectron. 25, 2592–2597. Lane, R.F., Blaha, C.D., 1990. Langmuir 6, 56–65. Li, G.D., Guo, C.L., Sun, C.H., Ju, Z.C., Yang, L.S., Xu, L.Q., Qian, Y.T., 2008. J. Phys. Chem. C 112, 1896–1900. Lin, K.C., Tsai, T.H., Chen, S.M., 2010. Biosens. Bioelectron 26, 608–614. Lin, L.Q., Chen, J.H., Yao, H., Chen, Y.Z., Zheng, Y.J., Lin, X.H., 2008a. Bioelectrochemistry 73, 11–17. Lin, L., Niu, H.J., Zhang, M.L., Song, W., Wang, Z., Bai, X.D., 2008b. Appl. Surf. Sci. 254, 7250–7254. Liu, J.W., Shao, M.W., Tang, Q., Chen, X.Y., Liu, Z.P., Qian, Y.T., 2003. Carbon 41, 1682–1685. Liu, A.H., Honma, I., Zhou, H.S., 2007. Biosens. Bioelectron. 23, 74–80. Ni, Y.B., Shao, M.W., Tong, Y.H., Qian, G.X., Wei, X.W., 2005. J. Solid State Chem. 178, 908–911. Nicholson, R.S., 1965. Anal. Chem. 37, 1351–1352. Poh, W.C., Loh, K.P., Zhang, W.D., Triparthy, S., Ye, J.S., Sheu, F.S., 2004. Langmuir 20, 5484–5492. Postma, A., Yan, Y., Wang, Y.Y., Zelikin, A.N., Tjipto, E., Caruso, F., 2009. Chem. Mater. 21, 3042–3044. Prasad, K.S., Muthuraman, G., Zen, J.M., 2008. Electrochem. Commun. 10, 559– 563. Safavi, A., Maleki, N., Moradlou, O., Tajabadi, F., 2006. Anal. Biochem. 359, 224– 229. Shakkthivel, P., Chen, S.M., 2007. Biosens. Bioelectron. 22, 1680–1687. Shao, Y.Y., Wang, X.Q., Engelhard, M., Wang, C.M., Dai, S., Liu, J., Yang, Z.G., Lin, Y.H., 2010. J. Power Sources 195, 4375–4379. Silva, R.P., Lima, A.W.O., Serrano, S.H.P., 2008. Anal. Chim. Acta 612, 89–98. Su, F.B., Zhao, X.S., Wang, Y., Wang, L.K., Lee, J.Y., 2006. J. Mater. Chem. 16, 4413–4419. Sun, X.M., Li, Y.D., 2004. Angew. Chem. Int. Ed. 43, 3827–3831. Tang, C.F., Kumar, S.A., Chen, S.M., 2008. Anal. Biochem. 380, 174–183. Thiagarajan, S., Tsai, T.H., Chen, S.M., 2009. Biosens. Bioelectron. 24, 2712–2715. Vinu, A., Sawant, D.P., Ariga, K., Hartmann, M., Halligudi, S.B., 2005. Microporous Mesoporous Mater. 80, 195–203. Wang, Z., Lin, J., Liang, Q., Wang, Y., Luo, G., 2002. Analyst 127, 653–658. Wang, Y.L., Nepal, D., Geckeler, K.E., 2005. J. Mater. Chem. 15, 1049–1054. Wen, Z.H., Wang, Q., Zhang, Q., Li, J.H., 2007. Electrochem. Commun. 9, 1867–1872. Weng, J., Xue, J.M., Wang, J., Ye, J.S., Cui, H.F., Sheu, F.S., Zhang, Q.Q., 2005. Adv. Funct. Mater. 15, 639–647. Wu, C.Z., Zhu, X., Ye, L.L., OuYang, C.Z., Hu, S.Q., Lei, L.Y., Xie, Y., 2006. Inorg. Chem. 45, 8543–8550. Xia, Y.D., Mokaya, R., 2004. Adv. Mater. 16, 886–891. Xia, Y.D., Yang, Z.X., Robert, M., 2004. J. Phys. Chem. B 108, 19293–19298. Xiao, Y.H., Guo, C.X., Li, C.M., Li, Y.B., Zhang, J., Xue, R.H., Zhang, S., 2007. Anal. Biochem. 371, 229–237. Xiong, Y.J., Xie, Y., Li, Z.Q., Wu, C.Z., Zhang, R., 2003. Chem. Commun., 904–905. Yogeswaran, U., Chen, S.M., 2007. Electrochim. Acta 52, 5985–5996. Zare, H.R., Rajabzadeh, N., Nasirizadeh, N., Ardakani, M., 2006. J. Electroanal. Chem. 589, 60–69. Zhang, M.N., Gong, K.P., Zhang, H.Z., Mao, L.Q., 2005. Biosens. Bioelectron. 20, 1270–1276. Zhang, R., Jin, G.D., Chen, D., Hu, X.Y., 2009. Sens. Actuators B 138, 174–181. Zhu, S.Y., Li, H.J., Niu, W.X., Xu, G.B., 2009. Biosens. Bioelectron. 25, 940–943.
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Hollow nitrogen-doped carbon microspheres pyrolyzed from self-polymerized dopamine and its application in simultaneous electrochemical determination of uric acid, ascorbic acid and dopamine Chunhui Xiao, Xiaochen Chu, Yan Yang, Xing Li, Xiaohua Zhang, Jinhua Chen ∗ State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Lushan Southern Road, Changsha 410082, PR China
a r t i c l e
i n f o
Article history: Received 23 August 2010 Received in revised form 26 November 2010 Accepted 26 November 2010 Available online 4 December 2010 Keywords: Hollow nitrogen-doped carbon microspheres Dopamine Ascorbic acid Uric acid Differential pulse voltammetry
a b s t r a c t Hollow nitrogen-doped carbon microspheres (HNCMS) as a novel carbon material have been prepared and the catalytic activities of HNCMS-modified glassy carbon (GC) electrode towards the electrooxidation of uric acid (UA), ascorbic acid (AA) and dopamine (DA) have also been investigated. Comparing with the bare GC and carbon nanotubes (CNTs) modified GC (CNTs/GC) electrodes, the HNCMS modified GC (HNCMS/GC) electrode has higher catalytic activities towards the oxidation of UA, AA and DA. Moreover, the peak separations between AA and DA, and DA and UA at the HNCMS/GC electrode are up to 212 and 136 mV, respectively, which are superior to those at the CNTs/GC electrode (168 and 114 mV). Thus the simultaneous determination of UA, AA and DA was carried out successfully. In the co-existence system of UA, AA and DA, the linear response range for UA, AA and DA are 5–30 M, 100–1000 M and 3–75 M, respectively and the detection limits (S/N = 3) are 0.04 M, 0.91 M and 0.02 M, respectively. Meanwhile, the HNCMS/GC electrode can be applied to measure uric acid in human urine, and may be useful for measuring abnormally high concentration of AA or DA. The attractive features of HNCMS provide potential applications in the simultaneous determination of UA, AA and DA. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Simultaneous determination of uric acid (UA), dopamine (DA) and ascorbic acid (AA) has received a great deal of attention (Xiao et al., 2007). Since UA, AA and DA are highly electrochemically active, electrochemical methods for their simultaneous determination have the inherent advantages of simplicity, rapidity, and high sensitivity. However, UA, AA and DA are oxidized at similar potentials with poor sensitivity at bare solid electrodes and the overlap of their voltammetric responses would confuse their simultaneous determination. In addition, the products of oxidation can be absorbed or electropolymerized onto the electrode surface, promoting its partial passivation and negatively affecting detectability and reusability of electrodes (Chen et al., 2009; Gonon et al., 1980; Lane and Blaha, 1990). To overcome these problems, various modified electrodes have been developed, such as polymer film modified electrodes (Cizewski and Milczarek, 1999; Gao and Huang, 1998; Kalimuthu and John, 2010; Lin et al., 2008a), nanoparticles modified electrodes (Huang et al., 2008; Shakkthivel and Chen, 2007; Tang et al., 2008;
∗ Corresponding author. Tel.: +86 731 88821818; fax: +86 731 88821818. E-mail addresses: [email protected], [email protected] (J. Chen). 0956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2010.11.041
Weng et al., 2005), electrochemically oxidized glassy carbon (GC) electrode (Thiagarajan et al., 2009), pyrolytic graphite electrode (Silva et al., 2008), screen-printed carbon electrode (Prasad et al., 2008), carbon ionic liquid electrode (Safavi et al., 2006), carbon nanohorn modified electrode (Zhu et al., 2009) and carbon nanotube modified electrodes (Deng et al., 2009; Hocevar et al., 2005; Kumar et al., 2010; Liu et al., 2007; Poh et al., 2004; Wang et al., 2002). Among these electrodes, carbon material based electrode is considered as one of the most effective electrodes to carry out the simultaneous determination of UA, AA and DA due to their remarkable electrocatalytic properties. On the other hand, hollow carbon microspheres (HCMS) with a structure of hollow cores and carbon shells have high specific surface area, low density, high surface permeability and good electronic properties, currently attract intense interest in drug delivery, active material encapsulation, lithium-ion battery, catalyst supports, hydrogen storage, damping materials, and templates for the synthesis of other useful hollow materials (Du et al., 2007; Gill and Ballesteros, 1998; Han et al., 2003; Huang et al., 1999; Su et al., 2006; Sun and Li, 2004; Wen et al., 2007). However, there are few works focused on their application in electrochemical biosensors. The synthesis methods used to produce HCMS include chemical vapor deposition (CVD), the catalytic-reduction route, the sol–gel method, the solid template approach and the solvothermal method
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(Dai et al., 2000; Kizukaa et al., 2009; Li et al., 2008; Ni et al., 2005; Xia and Mokaya, 2004; Xia et al., 2004; Xiong et al., 2003). It is noted that these methods are commonly carried out with multiple procedures or specific equipments. Other cases, such as the pyrolysis of the mixed carbon sources, usually led to the production of smaller proportion or less homogeneous HCMS (Liu et al., 2003). Based on the good features of HCMS and the deficiencies in the existing synthesis methods, we expect to find a simple, low-cost and effective approach to prepare HCMS and explore their application in simultaneous electrochemical determination of UA, AA and DA. It is well known that pyrolysis of core/shell polymer particles is a versatile method to obtain HCMS, whose pore size and shell thickness can be easily tailored by adjusting the core size and shell thickness of the core/shell polymers. Besides, it offers a simple way to introduce heteroatoms into carbon-based materials. Heteroatoms (such as nitrogen), which are present in the carbon materials, change the electron donor/acceptor characteristics of carbon depending on their chemical states, thus leading to an enhancement of electrochemical properties (Kima et al., 2008; Lin et al., 2008b; Shao et al., 2010). Herein, using DA as the source of carbon and nitrogen, we successfully synthesized the hollow nitrogen-doped carbon microspheres (HNCMS) by pyrolysis of polydopamine (PDA) wrapped SiO2 microspheres which were fabricated by the spontaneous selfpolymerization of dopamine onto silica microspheres. This method is versatile, relatively quick, and inexpensive. It was found that the HNCMS modified GC electrode had high electrochemical catalytic activities towards the oxidation of UA, AA and DA due to the hollow structure, good electrochemical properties and nitrogen-doping of the HNCMS. And in the co-existence system of UA, AA and DA, the simultaneous determination of UA, AA and DA could be simply achieved, which contributed greatly to the practical analysis of UA, AA and DA. 2. Experimental 2.1. Reagents Dopamine hydrochloride and SiO2 particles (diameter, ∼360 nm) were purchased from Alfa Aesar (USA) and used as received. Multi-walled carbon nanotubes (CNTs, >95%, diameter 20–40 nm, length 5–15 m) prepared by CVD were purchased from Shenzhen Nanotech Port Ltd. and purified by refluxing in concentrated nitric acid solution for 6 h. All other chemicals were of analytical grade. Aqueous solutions used throughout were prepared with ultra-pure water obtained from a Millipore system (>18 M cm). All electrochemical measurements were carried out at room temperature (25 ± 2 ◦ C). 2.2. Apparatus The scanning electron microscope (SEM) observations were conducted on JSM 6700F (JEOL, Japan). Nitrogen sorption isotherms and Brunauer–Emmett–Teller (BET) surface areas of the materials were determined by an ASAP 2010 Micrometrics sorptometer (America). Nitrogen content in HNCMS was determined by elemental analyzer (TCH-600, America). All electrochemical measurements were performed on a CHI 660D electrochemical workstation (Chenhua Instrument Company of Shanghai, China) with a conventional three-electrode system comprising a platinum wire as an auxiliary electrode, a saturated calomel electrode (SCE) as the reference electrode, and modified GC electrode as the working electrode. All the potentials in this paper were in respect to SCE.
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2.3. Preparation of HNCMS The preparation of HNCMS was composed of three steps and the schematic procedure for the synthesis of the HNCMS is shown in Scheme 1. PDA/SiO2 microspheres were synthesized according to the literature (Postma et al., 2009). In brief, 100 mg silica particles were washed thoroughly with 50 mM tris(hydroxymethyl)aminomethane (TRIS) buffer (pH = 8.5) and centrifuged three times, respectively. The deposit was resuspended in 40 mL 50 mM TRIS buffer (pH = 8.5) contained 2 mg mL−1 DA, followed by vigorous stirring for 24 h to form PDA/SiO2 microspheres (the molecular structure transformation during self-polymerization is shown in Scheme 1a). Then deep brown particles were centrifuged (3000 rpm, 1 min) and washed with fresh TRIS buffer (three times) to remove the tan colored solution, followed by drying in vacuum at 60 ◦ C overnight. The nitrogen-doped carbon/SiO2 microspheres were prepared by pyrolysis of the obtained PDA/SiO2 microspheres in a nitrogen atmosphere at 800 ◦ C for 2 h (Scheme 1b). Finally, the HNCMS were obtained via removal of the template (silica core) in 2 M HF + 8 M NH4 F solution at room temperature for 10 min, followed by successive centrifugation (13,000 rpm, 5 min) and washing three times, and drying in vacuum at 60 ◦ C overnight (Scheme 1c). The nitrogen content in the HNCMS was detected by elemental analyzer and is about 7.28 wt.%. 2.4. Preparation of HNCMS modified electrode Prior to use, GC electrodes (diameter = 3 mm) were carefully polished to a mirror-like plane with 0.5 and 0.05-m alumina slurries, successively. Afterward, the electrode was washed thoroughly with excess amounts of water and dried under nitrogen gas. The HNCMS modified GC (HNCMS/GC) electrode was prepared by casting 5 L HNCMS suspension (1 mg mL−1 HNCMS in N,Ndimethylformamide) on the surface of GC electrode and dried at room temperature for 24 h. For comparison, the CNTs/GC electrode was also prepared under the same procedure. For real sample analysis, no pretreatment process was performed. 25 L of the urine sample was diluted with 10 mL 0.1 M PBS (pH 7.0) and then the original concentration of uric acid in this diluted human urine was detected and is 14.16 M (Table S1, see the supplementary information). 3. Results and discussion 3.1. Characterization of HNCMS The morphology of the as-prepared samples was characterized by SEM. Fig. S1 (see the supplementary information) and Fig. 1 shows the SEM images of the SiO2 microspheres, PDA/SiO2 microspheres, carbon/SiO2 microspheres and HNCMS. From Fig. S1(A), it is noted that the diameter of SiO2 microspheres is about 360 nm. After 24 h self-polymerization of DA, PDA wrapped SiO2 microspheres with diameter of about 400 nm can be observed in Fig. S1(B) and the PDA shell is about 40 nm. Following carbonization in nitrogen atmosphere at 800 ◦ C for 2 h, the nitrogen-doped carbon/SiO2 composite is obtained. From Fig. S1(C), it is noted that the diameter of microsphere is decreased by ca. 5% due to the pyrolysis of PDA. Fig. 1 shows the SEM image of the HNCMS after removal of SiO2 template and the inset is the SEM image at higher magnification. The hollow feature of the microspheres with uniform diameter (∼380 nm) and shell thickness (∼20 nm) can be clearly observed. This indicates that structurally robust and replicated HNCMS can be fabricated successfully according to the procedure shown in Scheme 1.
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Scheme 1. Schematic routine for preparation of the hollow N-doped carbon microspheres.
Representative nitrogen adsorption/desorption isotherms and the corresponding Barrett–Joyner–Halenda (BJH) pore size distribution curve of the obtained HNCMS are shown in Fig. S2 (see the supplementary information). The nitrogen sorption isotherm of the product exhibits typical IV isotherm with H1 hysteresis at high relative pressure, which means that the obtained HNCMS possess mesoporous structure (Vinu et al., 2005). The pore-size distribution of HNCMS was calculated from nitrogen desorption using the BJH model, where the results show a distribution centered at 3.9 nm. The BET surface area of the HNCMS was calculated from the results of nitrogen adsorption and is 639.58 m2 g−1 , which is much higher than that of other HCMS (Wang et al., 2005; Wu et al., 2006) and CNTs (40–300 m2 g−1 ). Based on the hollow structure, high specific
Fig. 1. SEM image of hollow N-doped carbon microspheres. Inset: the magnified image on the corresponding area.
surface area and N-doping, HNCMS may have good electrochemical properties. 3.2. Electrochemical properties of HNCMS Fe(CN)6 3−/4− , as an electrochemical probe, is usually used to evaluate the electrochemical properties of the electrode. Fig. 2 shows cyclic voltammograms obtained at the bare GC, CNTs/GC and HNCMS/GC electrodes in 5 mM Fe(CN)6 3−/4− (1:1) + 0.1 M KCl solution. It is noted that the difference in potential between the anodic and cathodic peaks (Ep ) is 73 mV for the HNCMS/GC electrode, 85 mV for the CNTs/GC electrode and 110 mV for the bare GC electrode. As Ep is the function of the electron transfer rate, the
Fig. 2. CVs obtained at the bare GC (a), CNTs/GC (b) and HNCMS/GC (c) electrodes in 5 mM Fe(CN)6 3−/4− (1:1) + 0.1 M KCl aqueous solution. Scan rate: 50 mV s−1 .
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Fig. 3. (A) CVs at the bare GC (a1), CNTs/GC (b1) and HNCMS/GC electrodes (c1) in 0.1 M PBS containing 200 M UA. (B) CVs at the bare GC (d1), CNTs/GC (e1) and HNCMS/GC electrodes (f1) in 0.1 M PBS containing 1 mM AA. (C) CVs at the bare GC (g1), CNTs/GC (h1) and HNCMS/GC electrodes (i1) in 0.1 M PBS containing 100 M DA. Scan rate: 50 mV s−1 . Inset: the corresponding DPVs in 0.1 M PBS containing UA, AA and DA. (D) DPVs at the bare GC (j), CNTs/GC (k) and HNCMS/GC electrodes (l) in 0.1 M PBS containing 100 M UA + 500 M AA + 100 M DA.
lower Ep , the higher electron transfer rate. The electron transfer rate constant k0 for the ferricyanide system decreases with the increase of the value of Ep (Nicholson, 1965; Jia et al., 2007). Therefore, the order of the k0 value at different electrodes is as follows: HNCMS/GC > CNTs/GC > bare GC. Furthermore, the redox peak currents at the HNCMS/GC electrode are larger than that at the CNTs/GC and bare GC electrodes. This may result from the good electronic conductivity and large specific surface area of HNCMS. The smaller value of Ep and the higher redox peak currents indicate that the HNCMS/GC electrode has better electrochemical properties than the CNTs/GC and bare GC electrodes. The capability of electron transfer of different electrodes was further investigated by electrochemical impedance analysis and the corresponding results are shown in Fig. S3 (see the supplementary information). The order of the value of chargetransfer resistance (Rct ) for different electrodes is as follows: bare GC > CNTs/GC > HNCMS/GC. This result is in accordance with that observed in Fig. 2 and reconfirms that HNCMS possess good conductivity and electrochemical properties. These imply that HNCMS may be a promising electrochemical active material to construct electrochemical biosensors. 3.3. Electrocatalytic oxidation of UA, AA and DA Fig. 3 shows the cyclic voltammograms (CVs) of UA, AA and DA oxidation at the different electrodes and the inset plots show the corresponding differential pulse voltammetry (DPV) responses. For UA (200 M) oxidation, it can be seen from Fig. 3(A) that the oxidation peak potential at all of the electrodes appears at about 335 mV. However, the DPV peak current at the HNCMS/GC electrode (77.0 A) is 3.5 times and 22 times as high as that at the CNTs/GC electrode (22.0 A) and the bare GC electrode (3.5 A), respectively. The reversibility of the electrode process is not good although a weak reduction peak can be observed at the HNCMS/GC
electrode. Fig. 3(B) shows the electro-oxidation of 1 mM AA at the HNCMS/GC, CNTs/GC and bare GC electrodes. It is noted that AA oxidation occurs irreversibly with a large overpotential (326 mV) at the bare GC electrode, indicating the slow electron transfer because of the electrode fouling caused by the adsorption of oxidation products of AA (Zhang et al., 2005). Although both the oxidation potentials of AA at the HNCMS and CNTs modified GC electrodes shift negatively, oxidation potential at the HNCMS/GC electrode (−10 mV) is more negative than that at the CNTs/GC electrode (35 mV). Moreover, the DPV peak current at the HNCMS/GC electrode (39.8 A) shows 2 and 6 times higher than that at the CNTs/GC (19.8 A) and bare GC electrodes (6.6 A), respectively. In the case of AA, no reduction peak is observed at all electrodes, which show irreversible electrode processes for AA electro-oxidation. For 100 M DA (Fig. 3(C)), it can be found that a couple of the weak redox peaks with a Ep of 80 mV are observed at the bare GC electrode. The oxidation peak should result from the oxidation of dopamine to doaminequinone, while the reduction peak is caused by reducing doaminequinone to dopamine. However, a couple of the reversible and well-defined redox peaks with a Ep of 30 mV can be observed at both HNCMS/GC and CNTs/GC electrodes. In addition, the DPV peak current at the HNCMS/GC electrode (81.6 A) is 1.6 and 13.6 times higher than that at the CNTs/GC (49.6 A) and bare GC electrodes (6.0 A), respectively. These results indicate that the HNCMS/GC electrode possesses the best electrocatalytic properties towards the oxidation of UA, AA and DA among these three electrodes, especially negatively shift of the AA oxidation potential. This may be beneficial to simultaneous determination of UA, AA and DA with high sensitivity and excellent selectivity. In order to show clearly the ability of the electrode for the simultaneous determination of UA, AA and DA, the DPV results obtained in the co-existence system of UA, AA and DA are shown in Fig. 3(D). For the bare GC electrode, the oxidation peaks of AA
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and DA completely overlap and show a broad overlapped peak at 260 mV. The oxidation peak of UA appears at 392 mV and partially overlaps with that of AA and DA. At the CNTs/GC electrode, the DPV peak potentials are 296 mV, 14 mV, and 182 mV for UA, AA and DA, respectively. In contrast, the DPV peaks are well-resolved at the HNCMS/GC electrode with the peak potentials at about 292 mV, −56 mV, and 156 mV for UA, AA and DA, respectively. The peak separations for UA–DA and DA–AA at the HNCMS/GC electrode are up to 212 and 136 mV, respectively, which is superior to that at the CNTs/GC electrode (168 and 114 mV). These results reaffirm that HNCMS not only enhance the oxidation of UA, AA and DA, but also dramatically enlarge the peak separation among UA, AA and DA. The enlarged separation of the anodic peak potential, coupled with the increased sensitivity, indicates that the simultaneous determination of UA, AA and DA in the co-existence system is feasible. Also it is believed that the HNCMS modified electrode can be used for some interesting biological applications. 3.4. Determination of UA, AA and DA Since DPV has much higher current sensitivity and better resolution compared to CV, simultaneous determination of UA, AA and DA at HNCMS/GC electrode was carried out using DPV method. In ternary mixture, concentration of one species changed, and those of other two species remained constant. From Fig. 4(A), the peak current of UA in 0.1 M PBS containing 200 M AA and 40 M DA increases linearly with the increase of the UA concentration from 5 to 30 M. The corresponding linear function is Ip,UA (A) = 0.41 + 0.59 CUA (M) with a correlation coefficient of R = 0.9988 (relative standard deviations (R.S.D.) (n = 9) of responses for AA and DA are 1.7% and 3.3%, respectively). Similarly, the oxidation current of AA in 0.1 M PBS containing 80 M UA and 80 M DA increases with the increase of the AA concentration (Fig. 4(B)) and the linear range is 100–1000 M with the linear function Ip,AA (A) = 0.68 + 0.03 CAA (M) (R = 0.9989) (R.S.D. (n = 9) of responses for UA and DA are 2.3% and 2.6%, respectively). From Fig. 4(C), it can also be observed that the peak current of DA in 0.1 M PBS containing 80 M UA and 500 M AA increases linearly with the increase of the DA concentration from 3 to 70 M and the linear function is Ip,DA (A) = 1.14 + 0.93 CDA (M) with correlation of R = 0.9979 (R.S.D. (n = 10) of responses for UA and AA are 3.1% and 2.1%, respectively). The detection limits (S/N = 3) for UA, AA and DA are 0.04 M, 0.91 M and 0.02 M, respectively. Comparing the data shown in the previous literatures (Table 1), improved or comparable performance for the simultaneous determination of UA, AA and DA can be achieved using the HNCMS/GC electrode. Especially for AA, the detection limit by this method is improved mostly by more than one order of magnitude. To our best knowledge, direct electrochemical determination of AA in the co-existence system of UA, AA and DA with such a low detection limit is rarely reported. In order to investigate the stability of the electrode, the DPV responses in ternary mixture solution containing 80 M UA, 500 M AA and 80 M DA were recorded for 6 successive measurements. The current responses for six injections remained almost constant and the relative standard deviations (R.S.D.) of the same electrode were 2.0%, 0.7%, and 2.2% for UA, AA and DA, respectively. Also, there were no obvious decreases (3.1%, 1.8% and 3.7% for 80 M UA, 500 M AA and 80 M DA, respectively) in the responses of the HNCMS/GC electrode after its storage in PBS for a week. All those indicate that the stability of the HNCMS/GC electrode is good. Meanwhile, the analytical application of the HNCMS/GC electrode has been investigated by applying the HNCMS/GC electrode to determine UA, AA and DA in human urine by the calibration curve method, and the results are shown in Table S1 (see the supplementary information). The urine sample was diluted 400 times with 0.1 M PBS before the measurement to fit the calibration curve and
Fig. 4. DPVs at the HNCMS/GC electrode in 0.1 M PBS (A) containing 200 M AA, 40 M DA and different concentrations of UA (from inner to outer): 0, 5, 10, 20, 30, 50, 70, 110, 150 M; (B) containing 80 M UA, 80 M DA and different concentrations of AA (from inner to outer): 0, 100, 200, 300, 400, 500, 600, 800, 1000 M and (C) containing 80 M UA, 500 M AA and different concentrations of DA (from inner to outer): 0, 3, 6, 15, 25, 40, 55, 70, 100 and 150 M. Insets: plots of Ip vs. concentration for UA, AA and DA, respectively.
also reduce the matrix effect. No other pretreatment process was performed. It is noted that the spike recoveries of 105.2%, 97.8% and 99.5% for 10 M UA, 20 M DA and 200 M AA respectively are satisfactory, implying that the proposed electrochemical sensor (the HNCMS/GC electrode) may have great potential application for simultaneous determination of UA, AA and DA in real samples.
C. Xiao et al. / Biosensors and Bioelectronics 26 (2011) 2934–2939
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Table 1 Comparison of the analytical performance of the different modified electrodes for the simultaneous determination of AA, DA and UA. Electrode materials
Oracet blue f-MWNT PGE Oxidized GCE Poly-ACBK SWCNH CNT/PEDOT HNCMS a
Linear range (M)
LODa (M)
Sensitivity (A/M)
Reference
UA
AA
DA
UA
AA
DA
UA
AA
DA
20–600 75–180 2.5–30 1.97–9.88 1.0–120.0 0.06–10 10–250 3–30
40–800 75–187 25–500 197–988 50.0–1000 30–400 100–2000 100–1000
0.06–0.8 75–180 1–20 1.97–9.88 1.0–200.0 0.2–3.8 10–330 5–70
0.0071 0.08 1.52 0.0833 0.1311 5.95 – 0.59
0.0022 0.027 0.012 0.00009 0.0137 0.020 – 0.027
0.147 0.146 1.87 3.1575 0.1234 3.49 – 0.93
0.4 – 1.4
1.3 – 13
0.02 – 0.1
0.5 0.02 10 0.04
10.0 5 100 0.91
0.5 0.06 10 0.02
Zare et al. (2006) Yogeswaran and Chen (2007) Silva et al. (2008) Thiagarajan et al. (2009) Zhang et al. (2009) Zhu et al. (2009) Lin et al. (2010) This work
Limit of detection.
4. Conclusions Hollow nitrogen-doped carbon microspheres (HNCMS) were fabricated by pyrolysis of self-polymerized dopamine, and a novel and simple strategy for simultaneous determination of UA, AA and DA based on the HNCMS/GC electrode is presented. It was found that the electrochemical behaviors of UA, AA and DA have been improved at the HNCMS/GC electrode, which is ascribed to the unique hollow structure of HNCMS. Furthermore, compared with the CNTs modified GC electrode, the HNCMS/GC electrode shows more effective simultaneous determination of UA, AA and DA in coexistence system due to the larger separation of the oxidation peak potentials of UA, AA and DA. Additionally, the HNCMS/GC electrode has good stability. This study indicates a great promising of HNCMS in analytical applications. Acknowledgments This work was financially supported by the National Basic Research Program of China (No. 2009CB421601), NSFC (20975033, 20905024) and the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bios.2010.11.041. References Chen, P.Y., Vittal, R., Nien, P.C., Ho, K.C., 2009. Biosens. Bioelectron. 24, 3504–3509. Cizewski, A., Milczarek, G., 1999. Anal. Chem. 71, 1055–1061. Dai, K., Toshiaki, A., Yoshio, K., Akira, T., 2000. Chem. Mater. 12, 3397–3401. Deng, C.Y., Chen, J.H., Wang, M.D., Xiao, C.H., Nie, Z., Yao, S.Z., 2009. Biosens. Bioelectron. 24, 2091–2094. Du, H.D., Li, B.H., Kang, F.Y., Fu, R.W., Zeng, Y.Q., 2007. Carbon 45, 429–435. Gao, Z.Q., Huang, H., 1998. Chem. Commun., 2107–2108. Gill, I., Ballesteros, A., 1998. J. Am. Chem. Soc. 128, 8587–8592. Gonon, F., Buda, M., Cespuglio, R., Jouvert, M., Pajol, J., 1980. Nature 286, 902–904. Han, S.G., Yun, Y.K., Park, K.W., Sung, Y.E., Hyeon, T., 2003. Adv. Mater. 15, 1922–1925. Hocevar, S.B., Wang, J., Deo, R.P., Musameh, M., Ogorevc, B., 2005. Electroanalysis 17, 417–422. Huang, H.Y., Remsen, E.E., Kowalewski, T., Wooley, K.L., 1999. J. Am. Chem. Soc. 121, 3805–3806. Huang, J.S., Liu, Y., Hou, H.Q., You, T.Y., 2008. Biosens. Bioelectron. 24, 632–637. Jia, N.Q., Wang, Z.Y., Yang, G.F., Shen, H.B., Zhu, L.Z., 2007. Electrochem. Commun. 9, 233–238.
Kalimuthu, P., John, S.A., 2010. Talanta 80, 1686–1691. Kima, N.D., Kima, W., Joo, J.B., Oha, S., Kimb, P., Kimc, Y., Yi, J., 2008. J. Power Sources 180, 671–675. Kizukaa, T., Katoa, R., Miyazawab, K., 2009. Carbon 47, 138–144. Kumar, S.A., Wang, S.F., Yang, T.C.K., Yeh, C.T., 2010. Biosens. Bioelectron. 25, 2592–2597. Lane, R.F., Blaha, C.D., 1990. Langmuir 6, 56–65. Li, G.D., Guo, C.L., Sun, C.H., Ju, Z.C., Yang, L.S., Xu, L.Q., Qian, Y.T., 2008. J. Phys. Chem. C 112, 1896–1900. Lin, K.C., Tsai, T.H., Chen, S.M., 2010. Biosens. Bioelectron 26, 608–614. Lin, L.Q., Chen, J.H., Yao, H., Chen, Y.Z., Zheng, Y.J., Lin, X.H., 2008a. Bioelectrochemistry 73, 11–17. Lin, L., Niu, H.J., Zhang, M.L., Song, W., Wang, Z., Bai, X.D., 2008b. Appl. Surf. Sci. 254, 7250–7254. Liu, J.W., Shao, M.W., Tang, Q., Chen, X.Y., Liu, Z.P., Qian, Y.T., 2003. Carbon 41, 1682–1685. Liu, A.H., Honma, I., Zhou, H.S., 2007. Biosens. Bioelectron. 23, 74–80. Ni, Y.B., Shao, M.W., Tong, Y.H., Qian, G.X., Wei, X.W., 2005. J. Solid State Chem. 178, 908–911. Nicholson, R.S., 1965. Anal. Chem. 37, 1351–1352. Poh, W.C., Loh, K.P., Zhang, W.D., Triparthy, S., Ye, J.S., Sheu, F.S., 2004. Langmuir 20, 5484–5492. Postma, A., Yan, Y., Wang, Y.Y., Zelikin, A.N., Tjipto, E., Caruso, F., 2009. Chem. Mater. 21, 3042–3044. Prasad, K.S., Muthuraman, G., Zen, J.M., 2008. Electrochem. Commun. 10, 559– 563. Safavi, A., Maleki, N., Moradlou, O., Tajabadi, F., 2006. Anal. Biochem. 359, 224– 229. Shakkthivel, P., Chen, S.M., 2007. Biosens. Bioelectron. 22, 1680–1687. Shao, Y.Y., Wang, X.Q., Engelhard, M., Wang, C.M., Dai, S., Liu, J., Yang, Z.G., Lin, Y.H., 2010. J. Power Sources 195, 4375–4379. Silva, R.P., Lima, A.W.O., Serrano, S.H.P., 2008. Anal. Chim. Acta 612, 89–98. Su, F.B., Zhao, X.S., Wang, Y., Wang, L.K., Lee, J.Y., 2006. J. Mater. Chem. 16, 4413–4419. Sun, X.M., Li, Y.D., 2004. Angew. Chem. Int. Ed. 43, 3827–3831. Tang, C.F., Kumar, S.A., Chen, S.M., 2008. Anal. Biochem. 380, 174–183. Thiagarajan, S., Tsai, T.H., Chen, S.M., 2009. Biosens. Bioelectron. 24, 2712–2715. Vinu, A., Sawant, D.P., Ariga, K., Hartmann, M., Halligudi, S.B., 2005. Microporous Mesoporous Mater. 80, 195–203. Wang, Z., Lin, J., Liang, Q., Wang, Y., Luo, G., 2002. Analyst 127, 653–658. Wang, Y.L., Nepal, D., Geckeler, K.E., 2005. J. Mater. Chem. 15, 1049–1054. Wen, Z.H., Wang, Q., Zhang, Q., Li, J.H., 2007. Electrochem. Commun. 9, 1867–1872. Weng, J., Xue, J.M., Wang, J., Ye, J.S., Cui, H.F., Sheu, F.S., Zhang, Q.Q., 2005. Adv. Funct. Mater. 15, 639–647. Wu, C.Z., Zhu, X., Ye, L.L., OuYang, C.Z., Hu, S.Q., Lei, L.Y., Xie, Y., 2006. Inorg. Chem. 45, 8543–8550. Xia, Y.D., Mokaya, R., 2004. Adv. Mater. 16, 886–891. Xia, Y.D., Yang, Z.X., Robert, M., 2004. J. Phys. Chem. B 108, 19293–19298. Xiao, Y.H., Guo, C.X., Li, C.M., Li, Y.B., Zhang, J., Xue, R.H., Zhang, S., 2007. Anal. Biochem. 371, 229–237. Xiong, Y.J., Xie, Y., Li, Z.Q., Wu, C.Z., Zhang, R., 2003. Chem. Commun., 904–905. Yogeswaran, U., Chen, S.M., 2007. Electrochim. Acta 52, 5985–5996. Zare, H.R., Rajabzadeh, N., Nasirizadeh, N., Ardakani, M., 2006. J. Electroanal. Chem. 589, 60–69. Zhang, M.N., Gong, K.P., Zhang, H.Z., Mao, L.Q., 2005. Biosens. Bioelectron. 20, 1270–1276. Zhang, R., Jin, G.D., Chen, D., Hu, X.Y., 2009. Sens. Actuators B 138, 174–181. Zhu, S.Y., Li, H.J., Niu, W.X., Xu, G.B., 2009. Biosens. Bioelectron. 25, 940–943.