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Simultaneous electrochemical determination of guanosine and adenosine with graphene–ZrO2 nanocomposite modified carbon ionic liquid electrode
Biosensors and Bioelectronics 44 (2013) 146–151
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Simultaneous electrochemical determination of guanosine and adenosine with graphene–ZrO2 nanocomposite modified carbon ionic liquid electrode Wei Sun a,b,n, Xiuzhen Wang b, Xiaohuan Sun b, Ying Deng b, Jun Liu b, Bingxin Lei a, Zhenfan Sun a a
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 October 2012 Received in revised form 4 January 2013 Accepted 15 January 2013 Available online 29 January 2013
In this paper an ionic liquid 1-hexylpyridinium hexafluorophosphate based carbon ionic liquid electrode (CILE) was fabricated and used as the basal electrode, which was further modified by graphene (GR) and ZrO2 nanoparticle with chitosan (CTS) film to immobilize the nanocomposite. The modified electrode was denoted as CTS–GR–ZrO2/CILE and further used for the simultaneous detection of adenosine and guanosine. Electrochemical performances of the modified electrode were greatly enhanced due to the presence of GR–ZrO2 nanocomposite, and the direct electro-oxidation behaviors of adenosine and guanosine were carefully investigated. Both adenosine and guanosine exhibited an increase of the oxidation peak currents with the negative shift of the oxidation peak potentials on the modified electrode, which indicated the electrocatalytic activity of GR–ZrO2 nanocomposite on the electrode surface. Electrochemical parameters of adenosine and guanosine on CTS–GR–ZrO2/CILE were calculated respectively, and a new electroanalytical method for the simultaneous determination of adenosine and guanosine was further established with the peak-to-peak separation (DEp) as 0.225 V. The proposed method was successfully applied to detect adenosine and guanosine in human urine samples with satisfactory results. & 2013 Elsevier B.V. All rights reserved.
Keywords: Graphene ZrO2 nanoparticle Adenosine Guanosine Carbon ionic liquid electrode
1. Introduction As a two-dimensional (2D) carbon material which is comprised of sp2 hybridized single sheet carbon atoms, graphene (GR) has been widely investigated due to its unique physical and chemical properties, such as good thermal conductivity, high charge carrier mobility at room temperature and big specific surface area (Choi et al., 2010). Due to its specific electrochemical prosperities, GR and related materials have been used in different fields of electrochemistry including supercapacitor, Li-ion batteries, electrocatalysis and electrochemical sensors (Chen et al., 2010; Ratinac et al., 2011; Brownson and Banks, 2010). Hou et al. (2011) summarized the recent research and development of GR based electrochemical energy conversion and storage system. Liu et al. (2012) reviewed the emerging GR based sensors for the biological and chemical detection. Brownson et al. (2012) n Corresponding author at: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China. Tel./fax: þ 86 898 31381637. E-mail address: [email protected] (W. Sun).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.01.030
overviewed the fundamental concepts of GR electrochemistry and its prominent applications. Shan et al. (2010) applied ionic liquid (IL)-functionalized GR to construct an electrochemical biosensor for detection of NADH. Wu et al. (2010) proposed a chitosandispersed GR modified glassy carbon electrode (GCE) for the direct electron transfer of cytochrome C. Our group also applied GR composites modified electrode for the investigation on the protein electrochemistry (Ruan et al., 2012) and the detection of hydroquinone (Hu et al., 2012) or bisphenol A (Wang et al., 2012). So GR modified electrode has been devised for the electrochemical application. But GR nanosheets tend to aggregate back to graphite on the electrode surface, which limit its real application. In recent years GR based nanocomposites have been studied, which can modify the GR nanosheets and avoid the agglomeration. The presence of inorganic particles on the GR surface can not only prevent the restacking but also form a class of GR based nanocomposite with many new functions (Singh et al., 2011). Bai and Shen (2012) presented a review about the synthesis and application of GR–inorganic nanocomposites. ZrO2 nanoparticle is a commonly used inorganic oxide with good thermal stability, chemical inertness and lack of toxicity (Dobson and McQuillan,
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1997) and affinity for the groups containing oxygen (Fang et al., 1997), which has been used in the modified electrodes. Liu et al. (2004) investigated the direct electron transfer of hemoglobin on ZrO2 nanoparticle modified pyrolytic graphite electrode. Du et al. (2008) applied a ZrO2 nanoparticle modified electrode for the stripping voltammetric analysis of organophosphate pesticides. Zhu et al. (2004) fabricated an electrodeposited ZrO2 thin films modified gold electrode for electrochemical detection of DNA hybridization. Recently GR–ZrO2 nanocomposite had been synthesized by different methods. Yin et al. (2012) fabricated a GR–ZrO2 nanocomposite by simple mechanical mixing and pressureless sintering process, which resulted in a homogeneous and random distribution of GR in the ZrO2 matrix. Du et al. (2011) described a one-step electrodeposition method for GR–ZrO2 nanocomposite modified GCE and used for the detection of organophosphorus agents. Gong et al. (2012) also used a similar electrodeposition method for the synthesis of ZrO2 nanoparticles decorated GR hybrid nanosheets. The combination of GR–ZrO2 nanocomposite exhibits the individual characteristics of GR such as large surface area and good conductivity with that of ZrO2 nanoparticles such as biocompatibility and enrichment ability of oxygen groups, which has great potential applications in the field of electrochemical sensors. Purine nucleotides have exhibited many important metabolic and biological effects in human systems, and the concentration changes of these nucleosides in body fluids can be used to indicate various pathological changes such as carcinoma or liver disease (Yang et al., 2002), so it is necessary to establish sensitive methods for the nucleotides detection. Guanosine and adenosine are two important nucleosides that present in the molecular structure of nucleic acids, which are vital in various biological processes. For example, adenosine can modulate physiological functions in heart and brain, and regulate oxygen supply during cell stress and renal function (Kloor et al., 2000). Guanosine plays a protective role during brain ischemia (Frizzo et al., 2002) and mediates the process of RNA splicing (Piriev et al., 1998). Many analytical methods have been developed for the individual or simultaneous determination of guanosine and adenosine. Lin et al. (1997) achieved the simultaneous determination of ribonucleosides by capillary electrophoresis with a copper working electrode. Giannattasio et al. (2003) achieved simultaneous determination of purine nucleotides by ion-pair high performance liquid chromatography. Chen et al. (2008) reported an Ag–clad Au colloids film for the detection of adenosine using surface enhanced Raman scattering sensing platform based on a structure switching aptamer. Goyal et al. (2007) used a fullerene-C60 modified GCE for the voltammetric detection of adenosine and guanosine. In this work a GR–ZrO2 nanocomposite was fabricated and further modified on a 1-hexylpyridinium hexafluorophosphate (HPPF6) based carbon ionic liquid electrode (CILE). The modified electrode was further used for the detection of guanosine and adenosine. IL is a kind of green solvent with the characteristics such as high ionic conductivity, wide electrochemical windows and good solubility. Due to its specific properties IL had been used as the electrolyte or the modifier in the field of electrochemical sensor (Wei and Ivaska, 2008). CILE is a new type of working electrode that is prepared by using IL as the binder and the modifier in the traditional carbon paste electrode (CPE). Due to the presence of IL in the carbon paste, CILE had shown the advantages such as increased conductivity, easy preparation, good reversibility, high sensitivity and the ability to lower the overpotential of electroactive compounds (Maleki et al., 2006). Our group applied an N-butylpyridinium hexafluorophosphate modified CPE for the investigation on the electrochemical behaviors of guansoine (Sun et al., 2009), and simultaneously detection of adenosine and guanosine on 1-ethyl-3-methylimidazolium
147
ethylsulfate based CILE (Sun et al., 2011). The results showed that the modifier could promote the electrochemical performance with the simultaneous determination of guanosine and adenosine realized on the modified electrode. Due to the advantages of GR and ZrO2 nanoparticles and their synergistic effects, the GR–ZrO2 nanocomposite modified electrode was prepared and exhibited better electrochemical performances. Based on the electrochemical response of guanosine and adenosine on CTS–GR–ZrO2/CILE, a new electrochemical method was established for the simultaneous determination with good electrocatalytic activity, high sensitivity, good repeatability, long-term stability and low cost.
2. Experimental 2.1. Apparatus Cyclic voltammetry and differential pulse voltammetry were carried out on a CHI 1210A electrochemical workstation (Shanghai CH Instruments, China). Electrochemical impedance spectroscopy (EIS) was performed on a CHI 750B electrochemical workstation (Shanghai CH Instrument, China). A conventional three-electrode system was used with a CTS–GR–ZrO2/CILE (^ ¼4 mm) as working electrode, a saturated calomel electrode (SCE) as reference electrode and a platinum wire as auxiliary electrode. Scanning electron microscopy was performed on a JSM-6700F scanning electron microscope (Japan Electron Company, Japan). 2.2. Reagents Guanosine (99%, Sigma), adenosine (99%, Sigma), 1-hexylpyridinium hexafluorophosphate (HPPF6, Lanzhou Greenchem ILS. LICP. CAS., China) and graphite powder (average particle size of 30 mm, Shanghai Colloid Chemical Co., China) were used as received. Graphene oxide (GO) was synthesized according to the previous reports (Hummers and Offerman, 1958), which was further reduced to GR with the addition of hydrazine (Wang et al., 2009). The final blank GR were obtained by filtration and dried in vacuum. ZrO2 nanoparticle was prepared according to a reported method (Kim et al., 2009). 0.2 mol L 1 Britton–Robinson (B–R) buffer solutions with various pH values were used as the supporting electrolyte. Urine samples received from healthy laboratory personnel were used in the electrochemical measurement after 50 times dilution with B–R buffer solution. All the other chemicals were of analytical reagent grade and doubly distilled water was used in all the experiments. 2.3. Preparation of CTS–GR–ZrO2/CILE CILE was fabricated by mixing 0.8 g of HPPF6 and 1.6 g of graphite powder in a mortar and ground carefully. A portion of resulted homogeneous paste was packed firmly into a glass tube cavity (F ¼ 4 mm) and the electrical contact was established through a copper wire to the end of the paste in the inner hole of the tube. The surface of CILE was polished on a piece of polishing paper just before used. 0.5 mg of GR and 0.8 mg of ZrO2 nanoparticle were dispersed into 1.0 mL of 1.0% CTS solution (in 1.0% HAc) and ultrasonicated for 2 h to form a homogenous solution. Then 7.0 mL of CTS–GR–ZrO2 mixture was dropped on CILE surface and dried in the air. After the solvent was evaporated, the electrode was thoroughly rinsed with water and the modified electrode was noted as CTS–GR–ZrO2/CILE. For comparison CTS–ZrO2/CILE and CTS–GR/CILE were also fabricated with the similar procedure.
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2.4. Experimental procedure Electrochemical experiments were performed in 0.2 mol L 1 B–R buffer solution containing different concentrations of guanosine or adenosine or their mixtures. The preconcentration procedures were carried out at the potential of þ0.60 V for 300 s. After a 20 s of quiet period, cyclic voltammograms were recorded on CTS–GR–ZrO2/CILE at the scan rate of 100 mV s 1. Experimental parameters of differential pulse voltammetric method were set as: pulse amplitude 0.008 V, pulse width 0.05 s and pulse period 0.2 s.
3. Results and discussion 3.1. Characterization of CTS–GR–ZrO2/CILE The SEM images of GR nanosheets and GR–ZrO2 nanocomposite were observed with the results shown in Fig. 1. GR exhibited a typical wrinkle morphology and paper-like structure with single or very thin layers (Fig. 1A). It can be easy to distinguish the edges of individual sheet with wrinkled areas and well-packed film. When ZrO2 nanoparticle and GR were mixed together (Fig. 1B), it could be observed that ZrO2 nanoparticles with an average size of 60 nm were well-distributed on the surface of GR layers, indicating the uniform mixture of GR with ZrO2 nanoparticle. The decoration of ZrO2 nanoparticles on the GR surface can prevent the restacking of GR nanosheets and form a new kind of hybrid nanocomposite, which exhibits the synergistic effects from the individual components. Electrochemical impedance spectroscopy (EIS) is further used to record the impedance changes of the modified electrodes during the modification process with the results shown in Fig. 1C. The semicircle diameter of Nyquist plot reflects the electron transfer resistance (Ret), which is resulted from the electron transfer of the redox probe [Fe(CN)6]3 /4 . The Ret value of CILE (curve b) was got as 228 O and that of CTS–ZrO2/CILE (curve a) increased to 317 O, indicating the presence of CTS–ZrO2 film on the electrode surface hindered the electron transfer. While on CTS–GR/CILE (curve c) or CTS–GR–ZrO2/CILE (curve d) two almost straight lines appeared, which indicated that the Ret values were close to zero. The result can be attributed to the presence of higher conductive GR nanosheets on the electrode surface, which was benefit for the electron transfer with the interface conductivity and the surface area greatly improved.
3.2. Cyclic voltammograms of guanosine or adenosine Cyclic voltammograms of guanosine and adenosine on different modified electrodes were recorded with the results shown in Fig. 2. As for 100.0 mmol L 1 guanosine solution an irreversible
oxidation peak appeared without any reduction peak, indicating an irreversible electrode process. The result was similar to the reported mechanism of guanosine electro-oxidation (Sun et al., 2011). On CILE the oxidation peak located at 1.163 V with the oxidation peak current (Ip) as 2.97 10 5 A (curve a). On CTS–ZrO2/CILE the oxidation peak located at 1.154 V with the oxidation peak current as 7.56 10 5 A (curve b). The increase of the peak current and the decrease of peak potential are the key factors for an electrocatalytic process. So the presence of ZrO2 nanoparticles on CILE surface exhibited certain electrocatalytic activity. Nanosized ZrO2 has been used in the electrode modification with the ability to increase the effective surface area, which is benefit for the adsorption of the analyte (Du et al., 2008). On CTS–GR/CILE the oxidation peak appeared at 1.148 V with the peak current as 8.31 10 5 A (curve c), which further proved the electrocatalytic ability of GR on the electrode surface. GR has been elucidated with high surface area, good electrochemical conductivity and excellent electrocatalytic activity. Also the conductivity of GR is bigger than that of ZrO2 nanoparticles, so the electrochemical response of guanosine was bigger on CTS–GR/CILE than that of CTS–ZrO2/CILE. While on CTS–GR–ZrO2/CILE the oxidation peak potential and current were got as 1.122 V and 1.65 10 4 A (curve d). The smallest oxidation peak potential and the highest oxidation peak current indicated that the presence of GR–ZrO2 nanocomposite on the electrode surface exhibited the highest electrocatalytic activity. The results could be attributed the synergistic effects of the nanomaterials used. The similar results also appeared for 200.0 mmol L 1 adenosine on different modified electrodes. As shown in Fig. 2B, the oxidation peak potentials and peak currents on CILE (curve a), CTS–ZrO2/CILE (curve b), CTS–GR/ CILE (curve c) and CTS–GR–ZrO2/CILE (curve d) were got as 1.342 V, 1.322 V, 1.318 V, 1.303 V and 7.60 10 5 A, 1.57 10 4 A, 2.34 10 4 A and 3.38 10 4 A, respectively. The gradual changes of the electrochemical data further proved the functions of the nanomaterials present on the electrode surface, which exhibited excellent electrocatalytic activity with the decrease of the oxidation peak potential and the increase of the oxidation peak current. All the above results indicated that the presence of GR–ZrO2 nanocomposite on the modified electrode showed good electrocatalytic ability, which was attributed to the specific advantages of GR nanosheets such as high conductivity, fast electron transfer rate and big surface area, and that of ZrO2 nanoparticles with excellent biocompatibility. The simultaneous determination of guanosine and adenosine in the mixed solution was also achieved on CTS–GR–ZrO2/CILE. Fig. 2C showed the cyclic voltammograms of a mixture of 100.0 mmol L 1 guanosine and 200.0 mmol L 1 adenosine in 0.2 mol L 1 pH 5.7 B–R buffer solution on CTS–GR–ZrO2/CILE. Two oxidation peaks that appeared at 1.097 V and 1.322 V could be attributed to the oxidation of guanosine and adenosine, respectively. The peak-to-peak separation was calculated as 225 mV, which was bigger enough for the
Fig. 1. SEM images of (A) GR and (B) GR–ZrO2 nanocomposite, (C) EIS of (a) CTS–ZrO2/CILE, (b) CILE, (c) CTS–GR/CILE, (d) CTS–GR–ZrO2/CILE in the presence of a 10.0 mmol L 1 [Fe(CN)6]3 /4 and 0.1 mol L 1 KCl solution with the frequencies swept from 104 to 0.1 Hz.
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Fig. 2. (A) Cyclic voltammograms of 100.0 mmol L 1 guanosine on different electrodes in pH 6.0 B–R buffer solution; (B) cyclic voltammograms of 200.0 mmol L 1 adenosine on different electrodes in pH 5.5 B–R buffer solution (electrode from (a) to (d): CILE, CTS–ZrO2/CILE, CTS–GR/CILE, CTS–GR–ZrO2/CILE); (C) cyclic voltammograms of a mixture solution of 100.0 mmol L 1 guanosine and 200.0 mmol L 1 adenosine on CTS–GR–ZrO2/CILE in pH 5.7 B–R buffer solution (a) and that of the buffer solution (b).
simultaneous detection. So CTS–GR–ZrO2/CILE exhibited excellent distinguish ability for the simultaneous detection. 3.3. Effect of pH The effect of the buffer pH on the electrochemical responses were investigated by recording the cyclic voltammograms of 100.0 mmol L 1 purine nucleosides in B–R buffer solutions over the pH range from 3.0 to 8.0. The relationships of the oxidation peak potential and current with the buffer pH were further established. Both the oxidation peak potentials of guanosine and adenosine shifted linearly toward negative direction with the increase of the buffer pH, indicating that hydrogen ion took part in the electrode reaction. Two linear regression equations were calculated as Epa (V)¼ 0.043 pHþ1.284 (n ¼9, g ¼0.997) for guanosine and Epa (V)¼ 0.048 pH þ1.588 (n ¼9, g ¼0.995) for adenosine. The oxidation peak currents were also significantly affected by the buffer pH. Over the pH range studied, the maximal oxidation peak current of guanosine appeared at pH 6.0 and that of adenosine appeared at pH 5.5. Hence, the pH of buffer solution was fixed at 6.0 and 5.5 for the following individual investigation. As for the simultaneous determination pH 5.7 B–R buffer solution was selected as electrolyte to get the maximum sensitivity. 3.4. Optimization of the ratio of GR and ZrO2 nanoparticles The composition of the GR–ZrO2 nanocomposite will influence the electrochemical performance of the modified electrode, so the amount of GR and ZrO2 nanoparticles in the composite was investigated at different ratios. When the quantity of ZrO2 nanoparticles was too high, the interface conductivity decreased gradually, which may be due to the semiconductive ZrO2 nanoparticles and its aggregation to large particles. The electrochemical response reached the maximum when the mass ratio of 0.5 mg GR and 0.8 mg ZrO2 nanoparticles were mixed within 1.0 mL CTS solution, which was selected as the optimal ratio to prepare the nanocomposite for the electrode modification. 3.5. Effect of scan rate In order to study the electrochemical process of the purine nucleosides on CTS–GR–ZrO2/CILE, the effect of scan rate on the oxidation peak of 100.0 mmol L 1 purine nucleosides were
examined. Both the oxidation peak currents of guanosine and adenosine showed good linear relationships with scan rate in the range from 50 to 500 mV s 1, indicating an adsorption-controlled electrochemical process. The results indicated that the analytes were adsorbed on the electrode surface and then took place electron transfer with the electrode. The presence of GR–ZrO2 nanocomposite on the surface of CILE showed good adsorption ability to the analyte in the solution, which could be attributed to the increase of the surface area. The presence of ZrO2 nanoparticles on GR nanosheets can enlarge the sheet distances and result in a stable porous structure, which can adsorb more analytes in the composite. Then guanosine and adenosine can be adsorbed on the electrode surface and further exchange electrons with substrate electrode. The adsorbed amounts of guanosine and adenosine on the surface of CTS–GR–ZrO2/CILE were further calculated, respectively. Based on the following equation (Laviron, 1974): Ip ¼
nFQ u n2 F 2 AGT u ¼ 4RT 4RT
where n is the number of electron transferred, F (C mol 1) is the Faraday’s constant, A (cm2) is the effective area of the electrode, GT (mol cm 2) is the surface concentration of the electroactive matter, Q (C) is the quantity of charge consumed during the oxidation of the adsorbed guanosine or adenosine, u (mV s 1) is the scan rate. The linear relationship of the oxidation peak current with scan rate (u) was plotted with the equations as Ip (mA)¼1.2u (mV s 1)þ14.13 (n¼12, g ¼ 0.996) for guanosine and Ip (mA)¼2.8u (mV s 1)þ26.53 (n¼12, g ¼0.996) for adenosine. Based on the above equations the values of n and GT were calculated with the results as 1.88 and 2.81 10 9 mol cm 2 for guanosine, 2.17 and 5.91 10 9 mol cm 2 for adenosine, respectively. It can be seen that the value of n is similar with that reported by Goyal and Sangal (2002), ElNour and Brajter-Toth (2000) and Bi et al. (2005), indicating the similar electro-oxidation process. With the increase of scan rate the oxidation peak potential also moved to the positive direction. The linear dependence of Epa with ln u can be expressed with the equations as Epa (V)¼0.035ln u (V s 1)þ1.145 (n¼12, g ¼0.994) for guanosine and Epa (V)¼0.075ln u (V s 1)þ1.534 (n¼12, g ¼0.994) for adenosine. According to the Laviron’s equations (Laviron, 1974, 1979), the values of the charge transfer coefficient (a) and the electrode reaction standard rate constant (ks) were calculated with the results as 0.64 and 2.11 10 4 s 1 for guanosine, 0.72 and 9.33 10 5 s 1 for adenosine, respectively.
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3.6. Working curves for guanosine and adenosine The fabricated CTS–GR–ZrO2/CILE was further applied to guanosine and adenosine detection by the sensitive differential pulse voltammetry (DPV) with the typical voltammograms shown in Fig. 3 and the analytical parameters summarized in Table 1. As for the individual guanosine or adenosine solution, the oxidation peak current increased gradually with the increase of purine concentration (Fig. 3A for guanosine and Fig. 3B for adenosine). The detection limit was calculated as 0.123 mmol L 1 (3s) for guanosine and 0.131 mmol L 1 (3s) for adenosine. Fig. 3C shows the typical differential pulse voltammograms for the mixture solution of guanosine and adenosine with increasing concentrations. Two well-defined oxidation peaks appeared and were independent of each other without interferences, indicating the successfully simultaneous detection of guanosine and adenosine on CTS–GR–ZrO2/CILE. The detection limit for guanosine is lower than the reported results on that of boron doped diamond electrode (10.0 mmol L 1) (Fortin et al., 2004) and glassy carbon
Table 1 Analytical parameters on CTS–GR–ZrO2/CILE for the detection of guanosine and adenosine. Analytes
Concentration range (mmol L 1)
Guanosinea
0.4–80.0 80.0–200.0
Adenosine
a
0.4–20.0 20.0–360.0
Guanosineb
0.4–40.0 40.0–220.0
Adenosine
b
0.4–20.0 20.0–300.0
a b
Linear regression equations (Ip (mA), C (mmol L 1))
Ipa ¼ 0.246Cþ 2.46 (n¼ 12, g ¼0.993) Ipa ¼ 0.047Cþ 17.85 (n¼ 10, g ¼0.996) Ipa ¼ 0.44Cþ 5.43 (n¼ 11, g ¼0.995) Ipa ¼ 0.057Cþ 24.46 (n¼ 14, g ¼0.992) Ipa ¼ 0.277Cþ 2.97 (n¼ 16, g ¼0.993) Ipa ¼ 0.062Cþ 25.41 (n¼ 12, g ¼0.992) Ipa ¼ 0.476Cþ 6.36 (n¼ 11, g ¼0.995) Ipa ¼ 0.055Cþ 26.51 (n¼ 14, g ¼0.996)
Individual determination of guanosine or adenosine. Simultaneous determinations of guanosine and adenosine.
Detection limit (mmol L 1) (S/N ¼ 3) 0.123
0.131
0.117
electrode (0.2 mmol L 1) (Oliveira-Brett and Matysik, 1997). While the detection limit of adenosine is lower than that of fullerene-C60 modified GCE (0.302 mmol L 1) (Goyal et al., 2007) and carbon fiber ultra microelectrode (1.0 mmol L 1) (El-Nour and Brajter-Toth, 2000). So the proposed method showed the advantages including simple preparation procedure, higher sensitivity, lower detection limit and wide dynamic range for guanosine and adenosine detection. 3.7. Interferences The major interference for guanosine and adenosine determination comes from the common coexisting substances in biological fluids, which could lead to the appearance of either new voltammetric peaks or the overlap with the existing ones, and then influenced the voltammetric responses. The effects of 50.0 mmol L 1 of adrenaline, dopamine, uric acid and ascorbic acid on the voltammetric peak responses of the mixture for 50.0 mmol L 1 guanosine and 50.0 mmol L 1 adenosine were examined respectively. The results indicated that these substances exhibited less interference and no substantial changes appeared with the peak current responses changed below 75% for guanosine and adenosine. So the proposed electrode exhibited good selectivity in the electrochemical detection. 3.8. Analytical application CTS–GR–ZrO2/CILE was further utilized to detect guanosine and adenosine content in two human urine samples by the standard addition method. Prior to the analysis, the samples were diluted with pH 5.7 B–R buffer solution and the analytical results were presented in Table 2. The recoveries were determined by spiking the samples with a certain amount of standard solutions of adenosine and guanosine, and the results were found to be 95.56–102.91% for guanosine and 96.38–105.40% for adenosine. So this method could be applied to guanosine and adenosine detection in biological samples with satisfactory results. 3.9. Stability and reproducibility
0.144
The stability and reproducibility of CTS–GR–ZrO2/CILE was studied. The electrode was stored at room temperature when not in use, the initial response decreased for 2.2% and 4.3% after 15 days and 30 days storages, respectively, which indicated that the electrode had good stability. Furthermore, the fabrication
Fig. 3. Differential pulse voltammograms on CTS–GR–ZrO2/CILE with (A) different guanosine concentrations (from (a) to (h): 1.0, 5.0, 10.0, 30.0, 60.0, 80.0, 100.0, 200.0 mmol L 1); (B) different adenosine concentrations (from (a) to (h): 1.0, 4.0, 8.0, 20.0, 60.0, 100.0, 240.0, 360.0 mmol L 1); (C) the mixture of purine nucleosides solution (from (a) to (h): 0.8, 2.0, 6.0, 10.0, 40.0, 80.0, 100.0, 200.0 mmol L 1).
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Table 2 Detection of guanosine and adenosine concentration in human urine samples. Samples Added (mmol L 1)
Guanosine
Adenosine
Detected (mmol L 1)
Recovery (%)
Detected (mmol L 1)
Recovery (%)
1
10.0 40.0 80.0
9.87 38.67 82.33
98.70 96.68 102.91
9.66 42.16 78.75
96.60 105.40 98.44
2
10.0 40.0 80.0
10.12 39.04 76.45
101.20 97.60 95.56
9.92 38.55 82.06
99.20 96.38 102.58
reproducibility of ten electrodes made independently showed an acceptable reproducibility with the relative standard deviations (RSD) of 3.0570.13% for the detection of 100.0 mmol L 1 guanosine. Thus CTS–GR–ZrO2/CILE exhibited good stability and reproducibility for the electrochemical detection in general.
4. Conclusions In this paper a HPPF6 based CILE with GR–ZrO2 nanocomposite as modifier was fabricated and used for the investigation on the electrochemical behaviors of guanosine and adenosine. Remarkable enhancement of the oxidation peak currents were observed on the modified electrode with the negative shift of the oxidation peak potentials, indicating a typical electrocatalytic ability to the analyte. The results were attributed to the specific characteristics and synergistic effects of GR–ZrO2 nanocomposite presented on the electrode surface. The proposed method was further applied to the simultaneous detection of guanosine and adenosine in the mixture solution or the human urine samples with the good recovery, indicating the potential applications of GR–ZrO2 nanocomposite in the electrochemical sensor.
Acknowledgments We acknowledge the financial support of the National Natural Science Foundation of China (No. 21075071), the Natural Science Foundation of Hainan Province (212013) and the Foundation of Hainan Normal University. References Bai, S., Shen, X.P., 2012. RSC Advances 2, 64–98. Brownson, D.A.C., Banks, C.E., 2010. Analyst 135, 2768–2778. Brownson, D.A.C., Kampouris, D.K., Banks, C.E., 2012. Chemical Society Reviews 41, 6944–6976. Bi, S.P., Liu, B., Fan, F.F., Bard, A.J., 2005. Journal of American Chemical Society 127, 3690–3691.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Simultaneous electrochemical determination of guanosine and adenosine with graphene–ZrO2 nanocomposite modified carbon ionic liquid electrode Wei Sun a,b,n, Xiuzhen Wang b, Xiaohuan Sun b, Ying Deng b, Jun Liu b, Bingxin Lei a, Zhenfan Sun a a
Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 October 2012 Received in revised form 4 January 2013 Accepted 15 January 2013 Available online 29 January 2013
In this paper an ionic liquid 1-hexylpyridinium hexafluorophosphate based carbon ionic liquid electrode (CILE) was fabricated and used as the basal electrode, which was further modified by graphene (GR) and ZrO2 nanoparticle with chitosan (CTS) film to immobilize the nanocomposite. The modified electrode was denoted as CTS–GR–ZrO2/CILE and further used for the simultaneous detection of adenosine and guanosine. Electrochemical performances of the modified electrode were greatly enhanced due to the presence of GR–ZrO2 nanocomposite, and the direct electro-oxidation behaviors of adenosine and guanosine were carefully investigated. Both adenosine and guanosine exhibited an increase of the oxidation peak currents with the negative shift of the oxidation peak potentials on the modified electrode, which indicated the electrocatalytic activity of GR–ZrO2 nanocomposite on the electrode surface. Electrochemical parameters of adenosine and guanosine on CTS–GR–ZrO2/CILE were calculated respectively, and a new electroanalytical method for the simultaneous determination of adenosine and guanosine was further established with the peak-to-peak separation (DEp) as 0.225 V. The proposed method was successfully applied to detect adenosine and guanosine in human urine samples with satisfactory results. & 2013 Elsevier B.V. All rights reserved.
Keywords: Graphene ZrO2 nanoparticle Adenosine Guanosine Carbon ionic liquid electrode
1. Introduction As a two-dimensional (2D) carbon material which is comprised of sp2 hybridized single sheet carbon atoms, graphene (GR) has been widely investigated due to its unique physical and chemical properties, such as good thermal conductivity, high charge carrier mobility at room temperature and big specific surface area (Choi et al., 2010). Due to its specific electrochemical prosperities, GR and related materials have been used in different fields of electrochemistry including supercapacitor, Li-ion batteries, electrocatalysis and electrochemical sensors (Chen et al., 2010; Ratinac et al., 2011; Brownson and Banks, 2010). Hou et al. (2011) summarized the recent research and development of GR based electrochemical energy conversion and storage system. Liu et al. (2012) reviewed the emerging GR based sensors for the biological and chemical detection. Brownson et al. (2012) n Corresponding author at: Key Laboratory of Tropical Medicinal Plant Chemistry of Ministry of Education, College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China. Tel./fax: þ 86 898 31381637. E-mail address: [email protected] (W. Sun).
0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.01.030
overviewed the fundamental concepts of GR electrochemistry and its prominent applications. Shan et al. (2010) applied ionic liquid (IL)-functionalized GR to construct an electrochemical biosensor for detection of NADH. Wu et al. (2010) proposed a chitosandispersed GR modified glassy carbon electrode (GCE) for the direct electron transfer of cytochrome C. Our group also applied GR composites modified electrode for the investigation on the protein electrochemistry (Ruan et al., 2012) and the detection of hydroquinone (Hu et al., 2012) or bisphenol A (Wang et al., 2012). So GR modified electrode has been devised for the electrochemical application. But GR nanosheets tend to aggregate back to graphite on the electrode surface, which limit its real application. In recent years GR based nanocomposites have been studied, which can modify the GR nanosheets and avoid the agglomeration. The presence of inorganic particles on the GR surface can not only prevent the restacking but also form a class of GR based nanocomposite with many new functions (Singh et al., 2011). Bai and Shen (2012) presented a review about the synthesis and application of GR–inorganic nanocomposites. ZrO2 nanoparticle is a commonly used inorganic oxide with good thermal stability, chemical inertness and lack of toxicity (Dobson and McQuillan,
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1997) and affinity for the groups containing oxygen (Fang et al., 1997), which has been used in the modified electrodes. Liu et al. (2004) investigated the direct electron transfer of hemoglobin on ZrO2 nanoparticle modified pyrolytic graphite electrode. Du et al. (2008) applied a ZrO2 nanoparticle modified electrode for the stripping voltammetric analysis of organophosphate pesticides. Zhu et al. (2004) fabricated an electrodeposited ZrO2 thin films modified gold electrode for electrochemical detection of DNA hybridization. Recently GR–ZrO2 nanocomposite had been synthesized by different methods. Yin et al. (2012) fabricated a GR–ZrO2 nanocomposite by simple mechanical mixing and pressureless sintering process, which resulted in a homogeneous and random distribution of GR in the ZrO2 matrix. Du et al. (2011) described a one-step electrodeposition method for GR–ZrO2 nanocomposite modified GCE and used for the detection of organophosphorus agents. Gong et al. (2012) also used a similar electrodeposition method for the synthesis of ZrO2 nanoparticles decorated GR hybrid nanosheets. The combination of GR–ZrO2 nanocomposite exhibits the individual characteristics of GR such as large surface area and good conductivity with that of ZrO2 nanoparticles such as biocompatibility and enrichment ability of oxygen groups, which has great potential applications in the field of electrochemical sensors. Purine nucleotides have exhibited many important metabolic and biological effects in human systems, and the concentration changes of these nucleosides in body fluids can be used to indicate various pathological changes such as carcinoma or liver disease (Yang et al., 2002), so it is necessary to establish sensitive methods for the nucleotides detection. Guanosine and adenosine are two important nucleosides that present in the molecular structure of nucleic acids, which are vital in various biological processes. For example, adenosine can modulate physiological functions in heart and brain, and regulate oxygen supply during cell stress and renal function (Kloor et al., 2000). Guanosine plays a protective role during brain ischemia (Frizzo et al., 2002) and mediates the process of RNA splicing (Piriev et al., 1998). Many analytical methods have been developed for the individual or simultaneous determination of guanosine and adenosine. Lin et al. (1997) achieved the simultaneous determination of ribonucleosides by capillary electrophoresis with a copper working electrode. Giannattasio et al. (2003) achieved simultaneous determination of purine nucleotides by ion-pair high performance liquid chromatography. Chen et al. (2008) reported an Ag–clad Au colloids film for the detection of adenosine using surface enhanced Raman scattering sensing platform based on a structure switching aptamer. Goyal et al. (2007) used a fullerene-C60 modified GCE for the voltammetric detection of adenosine and guanosine. In this work a GR–ZrO2 nanocomposite was fabricated and further modified on a 1-hexylpyridinium hexafluorophosphate (HPPF6) based carbon ionic liquid electrode (CILE). The modified electrode was further used for the detection of guanosine and adenosine. IL is a kind of green solvent with the characteristics such as high ionic conductivity, wide electrochemical windows and good solubility. Due to its specific properties IL had been used as the electrolyte or the modifier in the field of electrochemical sensor (Wei and Ivaska, 2008). CILE is a new type of working electrode that is prepared by using IL as the binder and the modifier in the traditional carbon paste electrode (CPE). Due to the presence of IL in the carbon paste, CILE had shown the advantages such as increased conductivity, easy preparation, good reversibility, high sensitivity and the ability to lower the overpotential of electroactive compounds (Maleki et al., 2006). Our group applied an N-butylpyridinium hexafluorophosphate modified CPE for the investigation on the electrochemical behaviors of guansoine (Sun et al., 2009), and simultaneously detection of adenosine and guanosine on 1-ethyl-3-methylimidazolium
147
ethylsulfate based CILE (Sun et al., 2011). The results showed that the modifier could promote the electrochemical performance with the simultaneous determination of guanosine and adenosine realized on the modified electrode. Due to the advantages of GR and ZrO2 nanoparticles and their synergistic effects, the GR–ZrO2 nanocomposite modified electrode was prepared and exhibited better electrochemical performances. Based on the electrochemical response of guanosine and adenosine on CTS–GR–ZrO2/CILE, a new electrochemical method was established for the simultaneous determination with good electrocatalytic activity, high sensitivity, good repeatability, long-term stability and low cost.
2. Experimental 2.1. Apparatus Cyclic voltammetry and differential pulse voltammetry were carried out on a CHI 1210A electrochemical workstation (Shanghai CH Instruments, China). Electrochemical impedance spectroscopy (EIS) was performed on a CHI 750B electrochemical workstation (Shanghai CH Instrument, China). A conventional three-electrode system was used with a CTS–GR–ZrO2/CILE (^ ¼4 mm) as working electrode, a saturated calomel electrode (SCE) as reference electrode and a platinum wire as auxiliary electrode. Scanning electron microscopy was performed on a JSM-6700F scanning electron microscope (Japan Electron Company, Japan). 2.2. Reagents Guanosine (99%, Sigma), adenosine (99%, Sigma), 1-hexylpyridinium hexafluorophosphate (HPPF6, Lanzhou Greenchem ILS. LICP. CAS., China) and graphite powder (average particle size of 30 mm, Shanghai Colloid Chemical Co., China) were used as received. Graphene oxide (GO) was synthesized according to the previous reports (Hummers and Offerman, 1958), which was further reduced to GR with the addition of hydrazine (Wang et al., 2009). The final blank GR were obtained by filtration and dried in vacuum. ZrO2 nanoparticle was prepared according to a reported method (Kim et al., 2009). 0.2 mol L 1 Britton–Robinson (B–R) buffer solutions with various pH values were used as the supporting electrolyte. Urine samples received from healthy laboratory personnel were used in the electrochemical measurement after 50 times dilution with B–R buffer solution. All the other chemicals were of analytical reagent grade and doubly distilled water was used in all the experiments. 2.3. Preparation of CTS–GR–ZrO2/CILE CILE was fabricated by mixing 0.8 g of HPPF6 and 1.6 g of graphite powder in a mortar and ground carefully. A portion of resulted homogeneous paste was packed firmly into a glass tube cavity (F ¼ 4 mm) and the electrical contact was established through a copper wire to the end of the paste in the inner hole of the tube. The surface of CILE was polished on a piece of polishing paper just before used. 0.5 mg of GR and 0.8 mg of ZrO2 nanoparticle were dispersed into 1.0 mL of 1.0% CTS solution (in 1.0% HAc) and ultrasonicated for 2 h to form a homogenous solution. Then 7.0 mL of CTS–GR–ZrO2 mixture was dropped on CILE surface and dried in the air. After the solvent was evaporated, the electrode was thoroughly rinsed with water and the modified electrode was noted as CTS–GR–ZrO2/CILE. For comparison CTS–ZrO2/CILE and CTS–GR/CILE were also fabricated with the similar procedure.
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2.4. Experimental procedure Electrochemical experiments were performed in 0.2 mol L 1 B–R buffer solution containing different concentrations of guanosine or adenosine or their mixtures. The preconcentration procedures were carried out at the potential of þ0.60 V for 300 s. After a 20 s of quiet period, cyclic voltammograms were recorded on CTS–GR–ZrO2/CILE at the scan rate of 100 mV s 1. Experimental parameters of differential pulse voltammetric method were set as: pulse amplitude 0.008 V, pulse width 0.05 s and pulse period 0.2 s.
3. Results and discussion 3.1. Characterization of CTS–GR–ZrO2/CILE The SEM images of GR nanosheets and GR–ZrO2 nanocomposite were observed with the results shown in Fig. 1. GR exhibited a typical wrinkle morphology and paper-like structure with single or very thin layers (Fig. 1A). It can be easy to distinguish the edges of individual sheet with wrinkled areas and well-packed film. When ZrO2 nanoparticle and GR were mixed together (Fig. 1B), it could be observed that ZrO2 nanoparticles with an average size of 60 nm were well-distributed on the surface of GR layers, indicating the uniform mixture of GR with ZrO2 nanoparticle. The decoration of ZrO2 nanoparticles on the GR surface can prevent the restacking of GR nanosheets and form a new kind of hybrid nanocomposite, which exhibits the synergistic effects from the individual components. Electrochemical impedance spectroscopy (EIS) is further used to record the impedance changes of the modified electrodes during the modification process with the results shown in Fig. 1C. The semicircle diameter of Nyquist plot reflects the electron transfer resistance (Ret), which is resulted from the electron transfer of the redox probe [Fe(CN)6]3 /4 . The Ret value of CILE (curve b) was got as 228 O and that of CTS–ZrO2/CILE (curve a) increased to 317 O, indicating the presence of CTS–ZrO2 film on the electrode surface hindered the electron transfer. While on CTS–GR/CILE (curve c) or CTS–GR–ZrO2/CILE (curve d) two almost straight lines appeared, which indicated that the Ret values were close to zero. The result can be attributed to the presence of higher conductive GR nanosheets on the electrode surface, which was benefit for the electron transfer with the interface conductivity and the surface area greatly improved.
3.2. Cyclic voltammograms of guanosine or adenosine Cyclic voltammograms of guanosine and adenosine on different modified electrodes were recorded with the results shown in Fig. 2. As for 100.0 mmol L 1 guanosine solution an irreversible
oxidation peak appeared without any reduction peak, indicating an irreversible electrode process. The result was similar to the reported mechanism of guanosine electro-oxidation (Sun et al., 2011). On CILE the oxidation peak located at 1.163 V with the oxidation peak current (Ip) as 2.97 10 5 A (curve a). On CTS–ZrO2/CILE the oxidation peak located at 1.154 V with the oxidation peak current as 7.56 10 5 A (curve b). The increase of the peak current and the decrease of peak potential are the key factors for an electrocatalytic process. So the presence of ZrO2 nanoparticles on CILE surface exhibited certain electrocatalytic activity. Nanosized ZrO2 has been used in the electrode modification with the ability to increase the effective surface area, which is benefit for the adsorption of the analyte (Du et al., 2008). On CTS–GR/CILE the oxidation peak appeared at 1.148 V with the peak current as 8.31 10 5 A (curve c), which further proved the electrocatalytic ability of GR on the electrode surface. GR has been elucidated with high surface area, good electrochemical conductivity and excellent electrocatalytic activity. Also the conductivity of GR is bigger than that of ZrO2 nanoparticles, so the electrochemical response of guanosine was bigger on CTS–GR/CILE than that of CTS–ZrO2/CILE. While on CTS–GR–ZrO2/CILE the oxidation peak potential and current were got as 1.122 V and 1.65 10 4 A (curve d). The smallest oxidation peak potential and the highest oxidation peak current indicated that the presence of GR–ZrO2 nanocomposite on the electrode surface exhibited the highest electrocatalytic activity. The results could be attributed the synergistic effects of the nanomaterials used. The similar results also appeared for 200.0 mmol L 1 adenosine on different modified electrodes. As shown in Fig. 2B, the oxidation peak potentials and peak currents on CILE (curve a), CTS–ZrO2/CILE (curve b), CTS–GR/ CILE (curve c) and CTS–GR–ZrO2/CILE (curve d) were got as 1.342 V, 1.322 V, 1.318 V, 1.303 V and 7.60 10 5 A, 1.57 10 4 A, 2.34 10 4 A and 3.38 10 4 A, respectively. The gradual changes of the electrochemical data further proved the functions of the nanomaterials present on the electrode surface, which exhibited excellent electrocatalytic activity with the decrease of the oxidation peak potential and the increase of the oxidation peak current. All the above results indicated that the presence of GR–ZrO2 nanocomposite on the modified electrode showed good electrocatalytic ability, which was attributed to the specific advantages of GR nanosheets such as high conductivity, fast electron transfer rate and big surface area, and that of ZrO2 nanoparticles with excellent biocompatibility. The simultaneous determination of guanosine and adenosine in the mixed solution was also achieved on CTS–GR–ZrO2/CILE. Fig. 2C showed the cyclic voltammograms of a mixture of 100.0 mmol L 1 guanosine and 200.0 mmol L 1 adenosine in 0.2 mol L 1 pH 5.7 B–R buffer solution on CTS–GR–ZrO2/CILE. Two oxidation peaks that appeared at 1.097 V and 1.322 V could be attributed to the oxidation of guanosine and adenosine, respectively. The peak-to-peak separation was calculated as 225 mV, which was bigger enough for the
Fig. 1. SEM images of (A) GR and (B) GR–ZrO2 nanocomposite, (C) EIS of (a) CTS–ZrO2/CILE, (b) CILE, (c) CTS–GR/CILE, (d) CTS–GR–ZrO2/CILE in the presence of a 10.0 mmol L 1 [Fe(CN)6]3 /4 and 0.1 mol L 1 KCl solution with the frequencies swept from 104 to 0.1 Hz.
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149
Fig. 2. (A) Cyclic voltammograms of 100.0 mmol L 1 guanosine on different electrodes in pH 6.0 B–R buffer solution; (B) cyclic voltammograms of 200.0 mmol L 1 adenosine on different electrodes in pH 5.5 B–R buffer solution (electrode from (a) to (d): CILE, CTS–ZrO2/CILE, CTS–GR/CILE, CTS–GR–ZrO2/CILE); (C) cyclic voltammograms of a mixture solution of 100.0 mmol L 1 guanosine and 200.0 mmol L 1 adenosine on CTS–GR–ZrO2/CILE in pH 5.7 B–R buffer solution (a) and that of the buffer solution (b).
simultaneous detection. So CTS–GR–ZrO2/CILE exhibited excellent distinguish ability for the simultaneous detection. 3.3. Effect of pH The effect of the buffer pH on the electrochemical responses were investigated by recording the cyclic voltammograms of 100.0 mmol L 1 purine nucleosides in B–R buffer solutions over the pH range from 3.0 to 8.0. The relationships of the oxidation peak potential and current with the buffer pH were further established. Both the oxidation peak potentials of guanosine and adenosine shifted linearly toward negative direction with the increase of the buffer pH, indicating that hydrogen ion took part in the electrode reaction. Two linear regression equations were calculated as Epa (V)¼ 0.043 pHþ1.284 (n ¼9, g ¼0.997) for guanosine and Epa (V)¼ 0.048 pH þ1.588 (n ¼9, g ¼0.995) for adenosine. The oxidation peak currents were also significantly affected by the buffer pH. Over the pH range studied, the maximal oxidation peak current of guanosine appeared at pH 6.0 and that of adenosine appeared at pH 5.5. Hence, the pH of buffer solution was fixed at 6.0 and 5.5 for the following individual investigation. As for the simultaneous determination pH 5.7 B–R buffer solution was selected as electrolyte to get the maximum sensitivity. 3.4. Optimization of the ratio of GR and ZrO2 nanoparticles The composition of the GR–ZrO2 nanocomposite will influence the electrochemical performance of the modified electrode, so the amount of GR and ZrO2 nanoparticles in the composite was investigated at different ratios. When the quantity of ZrO2 nanoparticles was too high, the interface conductivity decreased gradually, which may be due to the semiconductive ZrO2 nanoparticles and its aggregation to large particles. The electrochemical response reached the maximum when the mass ratio of 0.5 mg GR and 0.8 mg ZrO2 nanoparticles were mixed within 1.0 mL CTS solution, which was selected as the optimal ratio to prepare the nanocomposite for the electrode modification. 3.5. Effect of scan rate In order to study the electrochemical process of the purine nucleosides on CTS–GR–ZrO2/CILE, the effect of scan rate on the oxidation peak of 100.0 mmol L 1 purine nucleosides were
examined. Both the oxidation peak currents of guanosine and adenosine showed good linear relationships with scan rate in the range from 50 to 500 mV s 1, indicating an adsorption-controlled electrochemical process. The results indicated that the analytes were adsorbed on the electrode surface and then took place electron transfer with the electrode. The presence of GR–ZrO2 nanocomposite on the surface of CILE showed good adsorption ability to the analyte in the solution, which could be attributed to the increase of the surface area. The presence of ZrO2 nanoparticles on GR nanosheets can enlarge the sheet distances and result in a stable porous structure, which can adsorb more analytes in the composite. Then guanosine and adenosine can be adsorbed on the electrode surface and further exchange electrons with substrate electrode. The adsorbed amounts of guanosine and adenosine on the surface of CTS–GR–ZrO2/CILE were further calculated, respectively. Based on the following equation (Laviron, 1974): Ip ¼
nFQ u n2 F 2 AGT u ¼ 4RT 4RT
where n is the number of electron transferred, F (C mol 1) is the Faraday’s constant, A (cm2) is the effective area of the electrode, GT (mol cm 2) is the surface concentration of the electroactive matter, Q (C) is the quantity of charge consumed during the oxidation of the adsorbed guanosine or adenosine, u (mV s 1) is the scan rate. The linear relationship of the oxidation peak current with scan rate (u) was plotted with the equations as Ip (mA)¼1.2u (mV s 1)þ14.13 (n¼12, g ¼ 0.996) for guanosine and Ip (mA)¼2.8u (mV s 1)þ26.53 (n¼12, g ¼0.996) for adenosine. Based on the above equations the values of n and GT were calculated with the results as 1.88 and 2.81 10 9 mol cm 2 for guanosine, 2.17 and 5.91 10 9 mol cm 2 for adenosine, respectively. It can be seen that the value of n is similar with that reported by Goyal and Sangal (2002), ElNour and Brajter-Toth (2000) and Bi et al. (2005), indicating the similar electro-oxidation process. With the increase of scan rate the oxidation peak potential also moved to the positive direction. The linear dependence of Epa with ln u can be expressed with the equations as Epa (V)¼0.035ln u (V s 1)þ1.145 (n¼12, g ¼0.994) for guanosine and Epa (V)¼0.075ln u (V s 1)þ1.534 (n¼12, g ¼0.994) for adenosine. According to the Laviron’s equations (Laviron, 1974, 1979), the values of the charge transfer coefficient (a) and the electrode reaction standard rate constant (ks) were calculated with the results as 0.64 and 2.11 10 4 s 1 for guanosine, 0.72 and 9.33 10 5 s 1 for adenosine, respectively.
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3.6. Working curves for guanosine and adenosine The fabricated CTS–GR–ZrO2/CILE was further applied to guanosine and adenosine detection by the sensitive differential pulse voltammetry (DPV) with the typical voltammograms shown in Fig. 3 and the analytical parameters summarized in Table 1. As for the individual guanosine or adenosine solution, the oxidation peak current increased gradually with the increase of purine concentration (Fig. 3A for guanosine and Fig. 3B for adenosine). The detection limit was calculated as 0.123 mmol L 1 (3s) for guanosine and 0.131 mmol L 1 (3s) for adenosine. Fig. 3C shows the typical differential pulse voltammograms for the mixture solution of guanosine and adenosine with increasing concentrations. Two well-defined oxidation peaks appeared and were independent of each other without interferences, indicating the successfully simultaneous detection of guanosine and adenosine on CTS–GR–ZrO2/CILE. The detection limit for guanosine is lower than the reported results on that of boron doped diamond electrode (10.0 mmol L 1) (Fortin et al., 2004) and glassy carbon
Table 1 Analytical parameters on CTS–GR–ZrO2/CILE for the detection of guanosine and adenosine. Analytes
Concentration range (mmol L 1)
Guanosinea
0.4–80.0 80.0–200.0
Adenosine
a
0.4–20.0 20.0–360.0
Guanosineb
0.4–40.0 40.0–220.0
Adenosine
b
0.4–20.0 20.0–300.0
a b
Linear regression equations (Ip (mA), C (mmol L 1))
Ipa ¼ 0.246Cþ 2.46 (n¼ 12, g ¼0.993) Ipa ¼ 0.047Cþ 17.85 (n¼ 10, g ¼0.996) Ipa ¼ 0.44Cþ 5.43 (n¼ 11, g ¼0.995) Ipa ¼ 0.057Cþ 24.46 (n¼ 14, g ¼0.992) Ipa ¼ 0.277Cþ 2.97 (n¼ 16, g ¼0.993) Ipa ¼ 0.062Cþ 25.41 (n¼ 12, g ¼0.992) Ipa ¼ 0.476Cþ 6.36 (n¼ 11, g ¼0.995) Ipa ¼ 0.055Cþ 26.51 (n¼ 14, g ¼0.996)
Individual determination of guanosine or adenosine. Simultaneous determinations of guanosine and adenosine.
Detection limit (mmol L 1) (S/N ¼ 3) 0.123
0.131
0.117
electrode (0.2 mmol L 1) (Oliveira-Brett and Matysik, 1997). While the detection limit of adenosine is lower than that of fullerene-C60 modified GCE (0.302 mmol L 1) (Goyal et al., 2007) and carbon fiber ultra microelectrode (1.0 mmol L 1) (El-Nour and Brajter-Toth, 2000). So the proposed method showed the advantages including simple preparation procedure, higher sensitivity, lower detection limit and wide dynamic range for guanosine and adenosine detection. 3.7. Interferences The major interference for guanosine and adenosine determination comes from the common coexisting substances in biological fluids, which could lead to the appearance of either new voltammetric peaks or the overlap with the existing ones, and then influenced the voltammetric responses. The effects of 50.0 mmol L 1 of adrenaline, dopamine, uric acid and ascorbic acid on the voltammetric peak responses of the mixture for 50.0 mmol L 1 guanosine and 50.0 mmol L 1 adenosine were examined respectively. The results indicated that these substances exhibited less interference and no substantial changes appeared with the peak current responses changed below 75% for guanosine and adenosine. So the proposed electrode exhibited good selectivity in the electrochemical detection. 3.8. Analytical application CTS–GR–ZrO2/CILE was further utilized to detect guanosine and adenosine content in two human urine samples by the standard addition method. Prior to the analysis, the samples were diluted with pH 5.7 B–R buffer solution and the analytical results were presented in Table 2. The recoveries were determined by spiking the samples with a certain amount of standard solutions of adenosine and guanosine, and the results were found to be 95.56–102.91% for guanosine and 96.38–105.40% for adenosine. So this method could be applied to guanosine and adenosine detection in biological samples with satisfactory results. 3.9. Stability and reproducibility
0.144
The stability and reproducibility of CTS–GR–ZrO2/CILE was studied. The electrode was stored at room temperature when not in use, the initial response decreased for 2.2% and 4.3% after 15 days and 30 days storages, respectively, which indicated that the electrode had good stability. Furthermore, the fabrication
Fig. 3. Differential pulse voltammograms on CTS–GR–ZrO2/CILE with (A) different guanosine concentrations (from (a) to (h): 1.0, 5.0, 10.0, 30.0, 60.0, 80.0, 100.0, 200.0 mmol L 1); (B) different adenosine concentrations (from (a) to (h): 1.0, 4.0, 8.0, 20.0, 60.0, 100.0, 240.0, 360.0 mmol L 1); (C) the mixture of purine nucleosides solution (from (a) to (h): 0.8, 2.0, 6.0, 10.0, 40.0, 80.0, 100.0, 200.0 mmol L 1).
W. Sun et al. / Biosensors and Bioelectronics 44 (2013) 146–151
Table 2 Detection of guanosine and adenosine concentration in human urine samples. Samples Added (mmol L 1)
Guanosine
Adenosine
Detected (mmol L 1)
Recovery (%)
Detected (mmol L 1)
Recovery (%)
1
10.0 40.0 80.0
9.87 38.67 82.33
98.70 96.68 102.91
9.66 42.16 78.75
96.60 105.40 98.44
2
10.0 40.0 80.0
10.12 39.04 76.45
101.20 97.60 95.56
9.92 38.55 82.06
99.20 96.38 102.58
reproducibility of ten electrodes made independently showed an acceptable reproducibility with the relative standard deviations (RSD) of 3.0570.13% for the detection of 100.0 mmol L 1 guanosine. Thus CTS–GR–ZrO2/CILE exhibited good stability and reproducibility for the electrochemical detection in general.
4. Conclusions In this paper a HPPF6 based CILE with GR–ZrO2 nanocomposite as modifier was fabricated and used for the investigation on the electrochemical behaviors of guanosine and adenosine. Remarkable enhancement of the oxidation peak currents were observed on the modified electrode with the negative shift of the oxidation peak potentials, indicating a typical electrocatalytic ability to the analyte. The results were attributed to the specific characteristics and synergistic effects of GR–ZrO2 nanocomposite presented on the electrode surface. The proposed method was further applied to the simultaneous detection of guanosine and adenosine in the mixture solution or the human urine samples with the good recovery, indicating the potential applications of GR–ZrO2 nanocomposite in the electrochemical sensor.
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