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Electrochemical sensor for simultaneous determination of uric acid, xanthine and hypoxanthine based on poly (bromocresol purple) modified glassy carbon electrode
Sensors and Actuators B 150 (2010) 43–49
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical sensor for simultaneous determination of uric acid, xanthine and hypoxanthine based on poly (bromocresol purple) modified glassy carbon electrode Yan Wang ∗ , Li-li Tong College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong 250014, China
a r t i c l e
i n f o
Article history: Received 26 April 2010 Received in revised form 8 July 2010 Accepted 27 July 2010 Available online 3 August 2010 Keywords: Bromocresol purple Chemical modified electrode Uric acid Xanthine Hypoxanthine
a b s t r a c t A novel electrochemical sensor based on electroactive-polymerized film of bromocresol purple (BCP) modified on glassy carbon electrode for simultaneous determination of uric acid (UA), xanthine (XA) and hypoxanthine (HX) was presented. The preparation and basic electrochemical performance of poly (BCP) film modified glassy carbon electrode were investigated firstly in details. The electrochemical behaviors of UA, XA and HX at the modified electrode were studied by cyclic voltammetry. The results showed that this new electrochemical sensor exhibited excellent electrocatalytic activity towards the oxidation of the three analytes. The anodic peaks of the three species were well defined with lowered oxidation potential and enhanced oxidation peak currents, so the poly (BCP) modified electrode was used for simultaneous voltammetric measurement of UA, XA and HX by differential pulse voltammetry. Under the optimum conditions, the calibration curves for UA, XA and HX were obtained over the range of 0.5–120, 0.1–100 and 0.2–80 mol L−1 , respectively. The detection limits for UA, XA and HX were 0.2, 0.06 and 0.12 mol L−1 , respectively. With good selectivity and sensitivity, the proposed method has been applied to simultaneous determination of UA, XA and HX in human serum with satisfactory results. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Uric acid (UA), xanthine (XA) and hypoxanthine (HX) are degradation products of purine metabolism in human beings and higher primates. UA is produced by xanthine oxidase from XA and HX, which in turn are produced from purine, so XA and HX are intermediates and UA is the final oxidation product of purine degradation metabolism. The three products can penetrate cell membranes and accumulate in extracellular fluids [1]. The concentration levels of them in body fluids such as human serum and urine are markers of many clinical conditions, including perinatal asphyxia, cerebral ischemia, hyperuricemia and gout, hence accurate detection and quantification of UA, XA, and HX in body fluids are critically important in study of the homeostasis of the xanthine oxidase system and clinical diagnosis at early stages of related diseases [2]. Current methods used to determine simultaneously the purine degradation products are high-performance liquid chromatography (HPLC) [3–5], capillary electrophoresis (CE) [6–8] and electrochemistry [9–16], in which HPLC methods require fastidious sample preparation, prolonged analysis time and expensive material, and CE methods need expensive apparatus.
∗ Corresponding author. Tel.: +86 531 89212269; fax: +86 531 88956828. E-mail address: [email protected] (Y. Wang). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.07.044
The development of electrochemical sensors through electrocatalysis for the determination of biologically important compounds is a major interest in current research [17,18]. Various biosensors based on the immobilized enzyme on electrode for determination of the three species has been reported [9,10], but these enzymatic methods are more expensive and lack stability and sensitivity. Non-enzymatic electrochemical sensors, on the other hand, are relatively sensitive, simple, cheap and rapid. It is even possible to determine the three compounds simultaneously. However, only a few reports appeared for the simultaneous determination of UA, XA and HX by non-enzymatic electrochemical approaches [11–16]. Yao et al. first studied the voltammetric oxidation of UA, XA and HX with a glassy carbon electrode, but the method suffered from serious interference by ascorbic acid and from nonlinear dependence of the current response on the concentration of these compounds [11]. Cai et al. proposed a method for the simultaneous differential pulse voltammetric determination of the three compounds with an electrochemically pretreated carbon paste electrode [12]. Zen et al. performed the simultaneous determination of HX, XA and UA using preanodized nontronite coated screen-printed carbon electrode (NSPE) [13], with detection limits of 0.34, 0.07 and 0.42 mol L−1 for them, respectively. Recently, Wang’s group developed an inlaying ultra-thin carbon paste electrode modified with functional single-wall carbon nanotubes (SWNTs/IUTCPE) for simultaneous determination of three
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purine derivatives [14]. Most recently, Kalimuthu and Abraham John described the simultaneous determination of ascorbic acid (AA), dopamine (DA), UA and XA using an ultra-thin electropolymerized film of 2-amino-1,3,4-thiadiazole (p-ATD) modified glassy carbon electrode [15]. Kumar’s group reported Ru (DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode for simultaneous electrochemical detection of HX, XA and UA in human urine [16]. In this paper, we prepared a new non-enzymatic electrochemical sensor based on electropolymerized film of bromocresol purple to catalyze the oxidation of UA, XA and HX, which provided a simple and sensitive voltammetric method for determining the concentrations of the three compounds simultaneously. Bromocresol purple (BCP), 5 ,5 -dibromo-o-cresolsulfophthalein is a pH indicator. It is also used as dye to measure albumin in medical laboratories. Its electropolymerization at the electrode surface and its function as an electrocatalyst have never been reported in the literature. In this work, we reported for the first time on a polymer film of BCP to modify glassy carbon electrode (GCE) and described the electrochemical behaviors of the novel poly (BCP) modified glassy carbon electrode. The poly (BCP) modified electrode possessed the larger real surface area, – conjugated bond, a great deal of active sites and better conductivity, which led to the dissimilar conjugation effect of the purine derivatives with the electrode interface. Therefore, the electrochemical reversibility of the oxidation of UA, XA and HX may be greatly improved in the presence of the poly (BCP) film by accelerating the rate of electron transfer, which indicated that the poly (BCP) film had excellent electrocatalytic activity for oxidation reactions of UA, XA and HX at the surface of the modified electrode. Moreover, the modified electrode showed good sensitivity, selectivity and reproducibility for the simultaneous determination of UA, XA and HX. Based on its excellent characteristics compared to other electrochemical sensors reported in terms of high sensitivity, wide linearity and good stability, the poly (BCP) modified electrode was satisfactorily used for the simultaneous determination of UA, XA and HX in human serum by differential pulse voltammetry (DPV).
2.3. Preparation of the poly (BCP) modified electrode Cyclic voltammetry (CV) was used to form polymerization film. Prior to its modification, the bare GCE was polished with 0.05 m ␣-alumina powder and rinsed with 1:1 HNO3 solution, ethanol, and doubly distilled water for 10 min successively. After the electrode was pretreated electrochemically by scanning in a 0.5 mol L−1 H2 SO4 solution between −0.5 and 1.5 V at 100 mV s−1 for ten times to get a stable background current, the BCP modified electrode was prepared by electropolymerization. The polymertic film was deposited by cyclic sweeping from −1.0 to 1.5 V at 100 mV s−1 for 20 cycles in 0.01 mol L−1 NaNO3 solution containing 2.0 × 10−3 mol L−1 BCP. After polymerization, the modified electrode was washed with doubly distilled water, and then air-dried. 2.4. Experimental methods Cyclic voltammetric and differential pulse voltammetric measurements were carried out with three electrodes in phosphate buffer solution. The cyclic voltammograms were recorded by cycling the potential between 0.0 and +1.4 V at a scan rate of 100 mV s−1 . The differential pulse voltammetric measurements were performed by applying a sweep potential from 0.0 to +1.2 V at pulse amplitude of 50 mV and pulse width of 0.1 s. All experiments were carried out at room temperature. The poly (BCP) modified electrode could be used repeatedly after rinsed with doubly distilled water and blotted with filter paper. 2.5. Sample preparation Blood samples were collected from healthy volunteers at the Hospital of Shandong Normal University. A 1.0 mL of fresh blood sample was obtained and centrifuged at 3000 rpm for 20 min to remove all precipitating materials. After the separated serum was diluted 20-fold with pH 6.5 phosphate buffer solution, an aliquot 10.0 mL of this test solution was transferred into the electrochemical cell to detect UA, XA and HX simultaneously by the proposed DPV method.
2. Experimental
3. Results and discussion
2.1. Chemicals
3.1. Preparation and characterization of electropolymerized BCP film at the GCE surface
UA, XA and HX were purchased from Sigma (USA). BCP and ascorbic acid were obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were of analytical grade and were used without further purification. The 0.067 mol L−1 phosphate buffer solutions (PBS) with various pH values were prepared by mixing the stock solutions of 0.067 mol L−1 KH2 PO4 and Na2 HPO4 . All solutions were prepared with doubly distilled water.
2.2. Apparatus Electrochemical measurements were performed with a LK2005 Microcomputer-based electrochemical system (LANLIKE, Tianjin, China). A conventional three-electrode cell was used, including a saturated calomel electrode (SCE) as reference electrode, a platinum sheet electrode as the counter electrode and a bare or modified glassy carbon disk electrode (GCE) with a diameter of 3.0 mm used as working electrode. All pH measurements were made with a pHS-3C digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass electrode. A KQ250B ultrasonic washer (Kunshan Ultrasonic Instrument Works, Kunshan, China) was used to wash the electrode.
Cyclic voltammetry was used to form the electropolymerization film. The potential scan range was the most important factor in preparing poly (BCP) film. If the positive potential value for polymerization was below 1.0 V or if the negative one was above −0.8 V, no polymer reaction occurred. The experimental result showed that the polymeric film formed was more conductive when the potential scan window was from −1.0 to 1.5 V. Therefore, it was selected as the electropolymerization potential window in this paper. Compared with other supporting electrolyte such as phosphate buffer solution used in electrodeposition process of polymertic film, the obtained polymertic film was more complete, uniform and compact, which showed better electrocatalytic activity to the oxidation of UA, XA and HX when NaNO3 was used as supporting electrolyte during polymer formation. Thus NaNO3 was chosen as supporting electrolyte for electropolymerization in this work. The consecutive cyclic voltammograms of 2.0 × 10−3 mol L−1 BCP in 0.01 mol L−1 NaNO3 solution at bare glassy carbon electrode were shown in Fig. 1. In the first cycle, one strong anodic peak was observed at 0.800 V(peak A), which might correspond to the oxidation of BCP monomer. A cathodic peak also appeared at about −0.480 V (peak B) in the first cycle. From the second cycle on, a new oxidation peak appeared with potential at 0.520 V (peak C)
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Fig. 2. Cyclic voltammograms of poly (BCP) modified glassy carbon electrode in pH 6.5 phosphate buffer solution at different scan rates. (a) 10 mV s−1 ; (b) 20 mV s−1 ; (c) 40 mV s−1 ; (d) 60 mV s−1 ; (e) 80 mV s−1 ; (f) 100 mV s−1 . Fig. 1. Cyclic voltammograms of BCP in electropolymerization process from 1 to 20 cycles. BCP: 2.0 × 10−3 mol L−1 ; supporting electrolyte: 0.01 mol L−1 NaNO3 ; scan rate: 100 mV s−1 .
in the cyclic voltammograms, and its peak current increased with successive scanning, indicating that film formation has occurred. During the polymerized process, peak A and peak B descended gradually with cyclic time increasing. Both the anodic and cathodic peak currents tended to be stable after 18 scans. These facts suggest that the initially formed poly (BCP) film had a leaching process with scan cycle increasing up to 18 times, which maybe implied a self-adjustment of the polymer film thickness at the GCE [19]. So the total number of the electropolymerizing scans was selected as twenty for forming stable polymer film. After modification, a uniform adherent blue polymer was formed on the GCE surface, which demonstrated that BCP was deposited on the surface of GCE by electropolymerization. The electrochemical behavior of the poly (BCP) modified electrode was studied in phosphate buffer solution by CV. The cyclic voltammograms of the poly (BCP) film recorded in pH 6.5 phosphate buffer solution at different scan rates were shown in Fig. 2, in which a chemically reversible redox couple was obtained in each cycle. The anodic peak current (ipa ) was linearly dependent on the scan rate () over the range of 10–100 mV s−1 , with the regression equation ipa (A) = 0.5596 + 0.04106 (mV s−1 ) (correlation coefficient, r = 0.9983), indicating a surface-controlled electrode processes. Moreover, the ratio of anodic peak current to cathodic peak current (ipa /ipc ) was almost equal to unity. With increasing scan rate, the separation of peak potentials (Ep = Epa − Epc ) would not be changed. The above results suggest that the electrode reaction is reversible [20]. Ep (=58.3 mV) is
close to 2.3RT/nF (or 59/n mV at 25 ◦ C), which was on accordance with a Nernst reversible behavior. So the number of transferred electrons in the electrode reaction was identified as one (n ≈ 1.01). The surface coverage of poly (BCP) modified electrode was made by adopting the method used by Sharp et al. [21]. According to this method, the peak current is related to the surface concentration of electroactive species, by the following equation: ip =
n2 F 2 A 4RT
where n represents the number of electrons involved in reaction (n = 1), A is the surface geometrical area of electrode (0.0706 cm2 ), represents the surface coverage concentration (mol cm−2 ) and other symbols have their usual meaning. From the slope of anodic peak currents versus scan rate, the surface concentration of poly (BCP) was calculated as 6.81 × 10−8 mol cm−2 . The result in Fig. 2 proved that BCP had been deposited on the surface of GCE by electropolymerization, and the functional group in the poly (BCP) film had good redox activity. The effect of pH on the electrochemical behavior of the poly (BCP) modified electrode was studied in phosphate buffer solution over the pH range from 4.5 to 9.2. The results showed that both Epa and Epc shifted towards the positive direction with increasing pH, which indicated that protons have taken part in the electrode processes. The anodic peak potentials exhibited a linear dependence on pH values with a slope of 56.4 mV pH−1 , which suggested that the ratio of the participated protons to the transferred electrons through the poly (BCP) film is 1:1. Thus, the possible reaction mechanism of poly (BCP) film on the glassy carbon electrode can be expressed as follows (Scheme 1): BCP (A) was first deposited at
Scheme 1. Mechanism of the poly (BCP) electrode reaction.
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Fig. 3. Cyclic voltammograms of the mixture containing UA, XA and HX at the bare glassy carbon electrode (A) and the poly (BCP) modified electrode (B) in pH 6.5 phosphate buffer solution. UA: 4.0 × 10−5 mol L−1 , XA: 2.0 × 10−5 mol L−1 , HX: 2.0 × 10−5 mol L−1 and scan rate: 100 mV s−1 .
the surface of GCE and oxidized to form a benzoquinone structure (B), then it was subsequently reduced to BCP at the reverse scan. The modified electrode exhibited a high stability when it was placed in dry state. No loss of electroactivity of the electrode was found for the continuously cyclical sweeping for 6 h. The modified electrode was also not deteriorated even for one month. 3.2. Electrochemical behaviors of UA, XA and HX at poly (BCP) modified electrode Fig. 3 demonstrated the CVs of the mixture solution containing UA, XA and HX at bare GCE and modified electrode in pH 6.5 PBS. As shown in Fig. 3A, all three compounds exhibited poor current responds and no well-defined peak of their oxidation could be seen at bare GCE, indicating a slow electron transfer kinetic. So it is impossible to use the bare electrode for simultaneous determination of the three species. In great contrast, when the modified electrode was used, the mixture displayed three well-defined and sensitive oxidation peaks. The peaks located at 0.321, 0.707 and 1.062 V, corresponding to the oxidation of UA, XA and HX, respectively. The differences of the oxidation peak potentials for UA–XA and XA–HX were 386 and 355 mV, respectively, which were enough large separations to allow the simultaneous determination of UA, XA and HX in a mixture. Meanwhile, it could be noticed that the peak currents of UA, XA and HX were enhanced significantly and all the three oxidation peak potentials had negative potential shifts at the poly (BCP) modified electrode. The enhanced current response and the lowered overpotential clearly indicated that poly (BCP) film can accelerate the rate of electron transfer and have excellent electrocatalytic activity towards the oxidation of UA, XA and HX. The poly (BCP) film has high concentration of the negatively charged surface functional group SO3 − and of the electron-rich O atom, which could interact with the purine derivatives, such as UA, XA and HX. Moreover, the poly (BCP) modified electrode possessed the larger real surface area, – conjugated bond, a great deal of active sites and better conductivity, which led to the dissimilar conjugation effect of the purine derivatives with the electrode interface. Therefore, the electrochemical reversibility of the oxidation of UA, XA and HX may be greatly improved in the presence of the poly (BCP) film by accelerating the rate of electron transfer. UA, XA and HX could be identified entirely at the poly
Fig. 4. Cyclic voltammograms of UA (A), XA (B) and HX (C) at modified electrode at different scan rates. (a) 20 mV s−1 , (b) 40 mV s−1 , (c) 60 mV s−1 , (d) 80 mV s−1 , (e) 100 mV s−1 , (f) 150 mV s−1 , (g) 200 mV s−1 , (h) 250 mV s−1 ; UA: 4.0 × 10−5 mol L−1 , XA: 2.0 × 10−5 mol L−1 , HX: 2.0 × 10−5 mol L−1 ; pH = 6.5 PBS. The inset shows the plot of dependence of ipa on .
(BCP) modified electrode based on its high electrocatalytic activity. 3.3. Effect of scan rate on the oxidation of UA, XA and HX at poly (BCP) modified electrode Fig. 4A–C showed the CVs of UA, XA and HX recorded at poly (BCP) modified electrode at different potential scan rates (), respectively. The plots of ipa as a function of for three molecules were also shown in the insets of Fig. 4. For all the three compounds, the oxidation peak current linearly increased with the scan rate, suggesting that the system presents features corresponding to an adsorption-controlled process for UA, XA and HX [22]. The linear regression equation relating ipa with the
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the oxidation reaction of the three species. The equation relating Epa with pH was found to be: Epa (V) = 0.7281 − 0.0618 pH (r = 0.9986) for UA, Epa (V) = 1.069 − 0.0556 pH (r = 0.9995) for XA, and Epa (V) = 1.468 − 0.0625 pH (r = 0.9979) for HX, respectively. The slopes of 61.8 mV pH−1 for UA, 55.6 mV pH−1 for XA and 62.5 mV pH−1 for HX are close to the theoretical value of 59 mV pH−1 , suggesting that the electron transfer step is preceded by a protonation with an equal number of protons and electrons involved in their oxidation. The effect of pH on anodic peak current for UA, XA and HX was shown in Fig. 5A. It could be seen that the anodic peak current of UA decreased with increasing of pH in a range of 4.5–9.2. However, the anodic peak current for XA and HX increased with increasing the pH up to 6.5, then the peak current decreased. The maximum value was observed at pH 6.5 for both XA and HX. 3.5. Simultaneous determination of UA, XA and HX
Fig. 5. Effects of pH on the anodic peak current (A) and anodic peak potential (B) of UA, XA and HX at the modified electrode, respectively. UA: 1.0 × 10−4 mol L−1 , XA: 4.0 × 10−5 mol L−1 , HX: 4.0 × 10−5 mol L−1 ; scan rate: 100 mV s−1 ; phosphate buffer solution.
scan rate over the range of 20–250 mV s−1 , was found to be: ipa (A) = 5.922 + 0.1261 (mV s−1 ) for UA with a correlation coefficient (r) of 0.9985, ipa (A) = 10.13 + 0.2228 (mV s−1 ) (r = 0.9991) for XA, and ipa (A) = 7.696 + 0.1395 (mV s−1 ) (r = 0.9982) for HX, respectively. In addition, it was observed that the catalytic oxidation peak potential (Epa ) shifted to more positive potentials with increasing scan rate for all the species. The analysis of these data showed that the plot of Epa versus the logarithm of scan rate presented a linear relation, indicating that the electrocatalytic oxidation of UA, XA and HX on the modified electrode surface is irreversible [20]. 3.4. Effect of pH on the oxidation of UA, XA and HX at poly (BCP) modified electrode Fig. 5 illustrated the dependence of the anodic peak potential (Epa ) and anodic peak current (ipa ) for UA (1.0 × 10−4 mol L−1 ), XA (4.0 × 10−5 mol L−1 ) and HX (4.0 × 10−5 mol L−1 ) on pH of buffer solution. The peak potentials (Epa ) for UA, XA and HX showed a same trend and shift almost linearly towards negative potentials when pH was increased in the range of 4.5–9.2, indicating that protons are directly involved in the rate determination step of
According to the above experimental results, the modified electrode showed good electrocatalytic activity for oxidation reaction of UA, XA and HX in wide pH range. The maximum peak current value can be observed at pH 4.5 for UA and pH 6.5 for both XA and HX, respectively. Because pH 6.5 is closer than pH 4.5 to the physiological pH value and the oxidation of the three compounds had high electrochemical response at this pH, it was selected as the optimum pH for the simultaneous determination of UA, XA and HX in this study. Since differential pulse voltammetry (DPV) has higher sensitivity and better resolution than cyclic voltammetry, DPV was used for simultaneous determination of UA, XA and HX. The DPV parameters were selected as pulse amplitude of 50 mV and pulse width of 100 ms for quantitative determination of three purine derivatives in order to obtain maximum peak current. The results showed that the peak currents of DPV are linearly proportional to the concentrations of the three compounds, respectively. Under the optimum conditions, the linear ranges and detection limits of UA, XA and HX were shown in Table 1, respectively. After every determination, the modified electrode can be regenerated by cycling between 0.0 and 1.4 V in a blank solution for six times to increase its reproducibility. To test the repeatability of the modified electrode, 2.0 × 10−5 mol L−1 of the three species were measured for six times. The relative standard deviations (RSD) of the peak currents for UA, XA and HX were 1.6%, 1.1% and 2.3%, respectively. The same solutions were also determined with six electrodes made independently, and the interelectrode RSD for peak currents UA, XA and HX were 2.2%, 1.5% and 2.8%, respectively. The above results demonstrated that the proposed method had high sensitivity, wide linear range and good repeatability. The comparison of this method with other electrochemical methods for the simultaneous determination of UA, XA and HX was listed in Table 2. 3.6. Interferences In order to check the intermolecular effects between UA, XA and HX, three different experiments were carried out under optimum conditions. In each experiment, the concentration of one of the three compounds was changed, whereas the concentrations
Table 1 Analytical characteristics for simultaneous determination of UA, XA and HX by the proposed method. Analyte
Linear range (mol L−1 )
UA XA HX
5.0 × 10−7 –1.2 × 10−4 1.0 × 10−7 –1.0 × 10−4 2.0 × 10−7 –8.0 × 10−5
Linear regression equation (i: A, C: mol L−1 ) ipa = 1.983 + 0.1612C ipa = 2.072 + 0.6456C ipa = 1.491 + 0.3892C
Correlation coefficient
Detection limit (mol L−1 )
0.9991 0.9995 0.9986
2.0 × 10−7 6.0 × 10−8 1.2 × 10−7
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Table 2 Comparison of the proposed method with other electrochemical methods for the simultaneous determination of UA, XA and HX. Linear range (mol L−1 )
Electrode
−8
Detection limit (mol L−1 ) −4
Ref.
−8
Electrochemically pretreated carbon paste electrode
UA: 5.94 × 10 –1.19 × 10 XA: 1.31 × 10−7 –6.57 × 10−4 HX: 3.67 × 10−7 –7.35 × 10−5
UA: 2.97 × 10 XA: 6.57 × 10−8 HX: 7.35 × 10−8
[12]
Preanodized nontronite coated screen-printed carbon electrode
UA: 2.0 × 10−6 –4.0 × 10−5 XA: 2.0 × 10−6 –4.0 × 10−5 HX: 4.0 × 10−6 –3.0 × 10−5
UA: 4.2 × 10−7 XA: 7.0 × 10−8 HX: 3.4 × 10−7
[13]
Inlaying ultra-thin carbon paste electrode modified with functional single-wall carbon nanotubes
UA: 1.0 × 10−7 –1.0 × 10−4 XA: 2.0 × 10−7 –1.0 × 10−4 HX: 8.0 × 10−7 –1.0 × 10−4
UA: 8.0 × 10−8 XA: 1.46 × 10−7 HX: 5.62 × 10−7
[14]
Poly (ATD) modified glassy carbon electrode
UA: 5.0 × 10−6 –4.5 × 10−5 XA: 5.0 × 10−6 –4.5 × 10−5
UA: 1.9 × 10−7 XA: 5.9 × 10−7
[15]
Ru (DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode
UA: 1.0 × 10−4 –7.0 × 10−4 XA: 5.0 × 10−5 –5.0 × 10−4 HX: 5.0 × 10−5 –3.0 × 10−4
UA: 3.72 × 10−7 XA: 2.35 × 10−6 HX: 2.37 × 10−6
[16]
Poly (BCP) modified glassy carbon electrode
UA: 5.0 × 10−7 –1.2 × 10−4 XA: 1.0 × 10−7 –1.0 × 10−4 HX: 2.0 × 10−7 –8.0 × 10−5
UA: 2.0 × 10−7 XA: 6.0 × 10−8 HX: 1.2 × 10−7
This work
Table 3 Determination of UA, XA and HX in human serum samples (n = 6). Sample
1 2 3
Original (mol L−1 )
Added (mol L−1 )
Found (mol L−1 )
Recovery (%)
UA
XA
HX
UA
XA
HX
UA
XA
HX
UA
XA
HX
11.89 8.10 22.45
1.23 5.45 5.86
3.68 1.89 –
10.00 8.0 20.0
2.0 6.0 6.0
4.0 2.0 10.0
22.18 15.86 41.85
3.19 11.72 11.65
7.47 3.81 9.93
101.3 98.5 98.6
98.8 102.4 98.2
97.3 97.9 99.3
of the other two species remained constant. The results in Fig. 6 showed that no obvious changes in the peak currents (10-fold of UA can decrease the peak currents of XA and HX in the ratio of 1.8% and 1.2%, respectively) and potentials of both XA and HX could be found while varying the concentration of UA. In addition, the oxidation peak current of UA increased linearly with a correlation coefficient of 0.9991 while increasing UA concentrations. Similarly, the oxidation peak current of XA or HX increased with increasing its concentrations while keeping the concentrations of the other two compounds constant, no interference can be observed for the
determination of XA or HX by the coexisting other two species. All the results identified that the oxidation processes of UA, XA and HX at poly (BCP) modified electrode are independent from each other, so it is possible to simultaneously determine the three compounds using the modified electrode in real samples without any interference with each other. For evaluating selectivity of the modified electrode, various possible interfering species were examined for their effect on the determination of UA, XA and HX (all of them were 2.0 × 10−5 mol L−1 ). The tolerance limit was taken as the maximum concentration of the foreign substances that caused an approximately ±5% relative error in the determination. If the tested coexisting interferents brought the detection current signal deviation below 5% under sufferable coexisting amount, we considered that the above substances have no interference. The results showed that 100-fold of glucose, lactic acid, serine, 60-fold of cysteine, ascorbic acid, 20-fold of uracil, theophylline, 10-fold of caffeine, albumin and 5-fold of dopamine, adenine, guanine, did not interfere with the determination of UA, XA and HX. From the experiments of the interference, it seems that the proposed method can be applicable to the detection of real biological samples.
3.7. Analytical applications
Fig. 6. Differential pulse voltammograms for UA with different concentrations in the presence of 10.0 mol L−1 XA and 20.0 mol L−1 HX at the poly (BCP) modified electrode. UA concentrations: (a) 20.0 mol L−1 , (b) 40.0 mol L−1 , (c) 60.0 mol L−1 , (d) 80.0 mol L−1 , (e) 100.0 mol L−1 ; pulse amplitude: 50 mV; pulse width: 100ms; pH = 6.5 PBS.
The practical analytical utility of the modified electrode was illustrated by simultaneous determination of UA, XA and HX in human serum samples. After all the serum samples were diluted 20-fold with pH 6.5 phosphate buffer solution, the three compounds were determined simultaneously by the proposed DPV method. The obtained results were summarized in Table 3. It can be seen that all spike recoveries were accurate and precise, which indicated the good applicability of the poly (BCP) modified electrode to simultaneous determination of UA, XA and HX in the real samples.
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4. Conclusions In this paper, the novel poly (BCP) modified electrode had been prepared by simple and fast electropolymerization method, and used as electrochemical sensor for the simultaneous determination of UA, XA and HX by DPV. The modified electrode had good stability, sensitivity and selectivity. The results demonstrated that the proposed method is a rapid, sensitive, and reproducible method for determination of purine derivatives in serum samples. Hence, we believe that the poly (BCP) modified electrode can become a useful tool for the assay of UA, XA and HX both in research assays and in clinical diagnosis due to its rapid speed, good precision and low cost of analysis. Acknowledgment The research presented in this manuscript was supported by Shandong Province Natural Science Foundation, China (No. Y2006B28). References [1] D.E. Metzler, Biochemistry: The Chemical Reactions of Living Cells, Academic Press, New York, 1977. [2] M. Heinig, R.J. Johnson, Role of uric acid in hypertension, renal disease, and metabolic syndrome, Clev. Clin. J. Med. 73 (2006) 1059–1064. [3] T. Yamamoto, Y. Moriwaki, S. Takahashi, Z. Tsutsumi, J. Yamakita, Y. Nasako, Determination of human plasma xanthine oxidase activity by highperformance liquid chromatography, J. Chromatogr. B: Biomed. Sci. Appl. 681 (1996) 395–400. [4] M. Czauderna, J. Kowalczyk, Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine by HPLC, J. Chromatogr. B: Biomed. Sci. Appl. 744 (2000) 129–138. [5] N. Cooper, R. Khosravan, C. Erdmann, J. Fiene, J.W. Lee, Quantification of uric acid, xanthine and hypoxanthine in human serum by HPLC for pharmacodynamic studies, J. Chromatogr. B: Biomed. Sci. Appl. 837 (2006) 1–10. [6] L. Terzuoli, B. Porcelli, C. Setacci, M. Guibbolini, Comparative determination of purine compounds in carotid plaque by capillary zone electrophoresis and high-performance liquid chromatography, J. Chromatogr. B: Biomed. Sci. Appl. 7289 (1999) 185–192. [7] M. Pizzichini, L. Arezzini, C. Billarelli, F. Carlucci, L. Terzuoli, Determination and separation of allantoin, uric acid, hypoxanthine, and xanthine by capillary zone electrophoresis, Adv. Exp. Med. Biol. 431 (1998) 797–800. [8] E. Caussˇıe, A. Pradelles, B. Dirat, A. Negre-Salvayre, R. Salvayre, F. Couderc, Simultaneous determination of allantoin, hypoxanthine, xanthine, and uric acid in serum/plasma by CE, Electrohporesis 28 (2007) 381–387. [9] M.A Carsol, G. Volpe, M. Mascini, Amperometric detection of uric acid and hypoxanthine with xanthine oxidase immobilized and carbon based screenprinted electrode: application for fish freshness determination, Talanta 44 (1997) 2151–2159.
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[10] M. Cubukcu, S. Timur, U. Anik, Examination of performance of glassy carbon paste electrode modified with gold nanoparticle and xanthine oxidase for xanthine and hypoxanthine detection, Talanta 74 (2007) 434–439. [11] T. Yao, Y. Taniguchi, T. Wasa, S. Musha, Anodic voltammetry and its analytical application to the detection and simultaneous determination of hypoxanthine, xanthine, and uric acid, Bull. Chem. Soc. Jpn. 51 (1978) 2937–2941. [12] X. Cai, K. Kalcher, C. Neuhold, Simultaneous determination of uric acid, xanthine and hypoxanthine with an electrochemically pretreated carbon paste electrode, Fresenius J. Anal. Chem. 348 (1994) 660–665. [13] J.M Zen, Y.Y. Lai, H.H. Yang, A. Senthil Kumar, Multianalyte sensor for the simultaneous determination of hypoxanthine, xanthine and uric acid based on a preanodized nontronite-coated screen-printed electrode, Sens. Actuators B: Chem. 84 (2002) 237–244. [14] Z.H. Wang, X.Y. Dong, J. Li, An inlaying ultra-thin carbon paste electrode modified with functional single-wall carbon nanotubes for simultaneous determination of three purine derivatives, Sens. Actuators B: Chem. 131 (2008) 411–416. [15] P. Kalimuthu, S. Abraham John, Simultaneous determination of ascorbic acid, dopamine, uric acid and xanthine using a nanostructured polymer film modified electrode, Talanta 80 (2010) 1686–1691. [16] A.S. Kumar, P. Swetha, Ru(DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode for simultaneous electrochemical detection of hypoxanthine, xanthine and uric acid, J. Electroanal. Chem. 642 (2010) 135–142. [17] Y. Wang, Z.Z. Chen, A novel poly(taurine) modified glassy carbon electrode for the simultaneous determination of epinephrine and dopamine, Colloid Surf. B 74 (2009) 322–327. [18] Y. Wang, Z.Z. Chen, A novel poly(cyanocobalamin) modified glassy carbon electrode as electrochemical sensor for voltammetric determination of peroxynitrite, Talanta 82 (2010) 534–539. [19] H. Yao, Y. Sun, X. Lin, Y. Tang, L. Huang, Electrochemical characterization of poly(eriochrome black T) modified glassy carbon electrode and its application to simultaneous determination of dopamine, ascorbic acid and uric acid, Electrochim. Acta 52 (2007) 6165–6171. [20] A.P. Brown, F.C. Anson, Cyclic and differential pulse voltammetric behavior of reactants confined to the electrode surface, Anal. Chem. 49 (1977) 1589– 1595. [21] M. Sharp, M. Petersson, K. Edstrom, General expression of the linear potential sweep voltammogram for a surface redox reaction with interactions between the adsorbed molecules: applications to modified electrodes, J. Electroanal. Chem. 95 (1979) 123–130. [22] A.J. Bard, L.R. Faulkner, Electrochemical Methods: Fundamentals and Applications, second ed, Wiley, New York, 2000.
Biographies Yan Wang received her master degree in analytical chemistry in 1995 from Shandong University, PR China. She is currently an associate professor in the Department of Chemistry at Shandong Normal University, PR China. Her research interests include electroanalytical chemistry, bioanalytical chemistry, and capillary electrophoresis. Li-li Tong is a lecturer in the Department of Chemistry at Shandong Normal University, PR China. Her research area is bioanalytical chemistry.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Electrochemical sensor for simultaneous determination of uric acid, xanthine and hypoxanthine based on poly (bromocresol purple) modified glassy carbon electrode Yan Wang ∗ , Li-li Tong College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, Shandong 250014, China
a r t i c l e
i n f o
Article history: Received 26 April 2010 Received in revised form 8 July 2010 Accepted 27 July 2010 Available online 3 August 2010 Keywords: Bromocresol purple Chemical modified electrode Uric acid Xanthine Hypoxanthine
a b s t r a c t A novel electrochemical sensor based on electroactive-polymerized film of bromocresol purple (BCP) modified on glassy carbon electrode for simultaneous determination of uric acid (UA), xanthine (XA) and hypoxanthine (HX) was presented. The preparation and basic electrochemical performance of poly (BCP) film modified glassy carbon electrode were investigated firstly in details. The electrochemical behaviors of UA, XA and HX at the modified electrode were studied by cyclic voltammetry. The results showed that this new electrochemical sensor exhibited excellent electrocatalytic activity towards the oxidation of the three analytes. The anodic peaks of the three species were well defined with lowered oxidation potential and enhanced oxidation peak currents, so the poly (BCP) modified electrode was used for simultaneous voltammetric measurement of UA, XA and HX by differential pulse voltammetry. Under the optimum conditions, the calibration curves for UA, XA and HX were obtained over the range of 0.5–120, 0.1–100 and 0.2–80 mol L−1 , respectively. The detection limits for UA, XA and HX were 0.2, 0.06 and 0.12 mol L−1 , respectively. With good selectivity and sensitivity, the proposed method has been applied to simultaneous determination of UA, XA and HX in human serum with satisfactory results. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Uric acid (UA), xanthine (XA) and hypoxanthine (HX) are degradation products of purine metabolism in human beings and higher primates. UA is produced by xanthine oxidase from XA and HX, which in turn are produced from purine, so XA and HX are intermediates and UA is the final oxidation product of purine degradation metabolism. The three products can penetrate cell membranes and accumulate in extracellular fluids [1]. The concentration levels of them in body fluids such as human serum and urine are markers of many clinical conditions, including perinatal asphyxia, cerebral ischemia, hyperuricemia and gout, hence accurate detection and quantification of UA, XA, and HX in body fluids are critically important in study of the homeostasis of the xanthine oxidase system and clinical diagnosis at early stages of related diseases [2]. Current methods used to determine simultaneously the purine degradation products are high-performance liquid chromatography (HPLC) [3–5], capillary electrophoresis (CE) [6–8] and electrochemistry [9–16], in which HPLC methods require fastidious sample preparation, prolonged analysis time and expensive material, and CE methods need expensive apparatus.
∗ Corresponding author. Tel.: +86 531 89212269; fax: +86 531 88956828. E-mail address: [email protected] (Y. Wang). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.07.044
The development of electrochemical sensors through electrocatalysis for the determination of biologically important compounds is a major interest in current research [17,18]. Various biosensors based on the immobilized enzyme on electrode for determination of the three species has been reported [9,10], but these enzymatic methods are more expensive and lack stability and sensitivity. Non-enzymatic electrochemical sensors, on the other hand, are relatively sensitive, simple, cheap and rapid. It is even possible to determine the three compounds simultaneously. However, only a few reports appeared for the simultaneous determination of UA, XA and HX by non-enzymatic electrochemical approaches [11–16]. Yao et al. first studied the voltammetric oxidation of UA, XA and HX with a glassy carbon electrode, but the method suffered from serious interference by ascorbic acid and from nonlinear dependence of the current response on the concentration of these compounds [11]. Cai et al. proposed a method for the simultaneous differential pulse voltammetric determination of the three compounds with an electrochemically pretreated carbon paste electrode [12]. Zen et al. performed the simultaneous determination of HX, XA and UA using preanodized nontronite coated screen-printed carbon electrode (NSPE) [13], with detection limits of 0.34, 0.07 and 0.42 mol L−1 for them, respectively. Recently, Wang’s group developed an inlaying ultra-thin carbon paste electrode modified with functional single-wall carbon nanotubes (SWNTs/IUTCPE) for simultaneous determination of three
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purine derivatives [14]. Most recently, Kalimuthu and Abraham John described the simultaneous determination of ascorbic acid (AA), dopamine (DA), UA and XA using an ultra-thin electropolymerized film of 2-amino-1,3,4-thiadiazole (p-ATD) modified glassy carbon electrode [15]. Kumar’s group reported Ru (DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode for simultaneous electrochemical detection of HX, XA and UA in human urine [16]. In this paper, we prepared a new non-enzymatic electrochemical sensor based on electropolymerized film of bromocresol purple to catalyze the oxidation of UA, XA and HX, which provided a simple and sensitive voltammetric method for determining the concentrations of the three compounds simultaneously. Bromocresol purple (BCP), 5 ,5 -dibromo-o-cresolsulfophthalein is a pH indicator. It is also used as dye to measure albumin in medical laboratories. Its electropolymerization at the electrode surface and its function as an electrocatalyst have never been reported in the literature. In this work, we reported for the first time on a polymer film of BCP to modify glassy carbon electrode (GCE) and described the electrochemical behaviors of the novel poly (BCP) modified glassy carbon electrode. The poly (BCP) modified electrode possessed the larger real surface area, – conjugated bond, a great deal of active sites and better conductivity, which led to the dissimilar conjugation effect of the purine derivatives with the electrode interface. Therefore, the electrochemical reversibility of the oxidation of UA, XA and HX may be greatly improved in the presence of the poly (BCP) film by accelerating the rate of electron transfer, which indicated that the poly (BCP) film had excellent electrocatalytic activity for oxidation reactions of UA, XA and HX at the surface of the modified electrode. Moreover, the modified electrode showed good sensitivity, selectivity and reproducibility for the simultaneous determination of UA, XA and HX. Based on its excellent characteristics compared to other electrochemical sensors reported in terms of high sensitivity, wide linearity and good stability, the poly (BCP) modified electrode was satisfactorily used for the simultaneous determination of UA, XA and HX in human serum by differential pulse voltammetry (DPV).
2.3. Preparation of the poly (BCP) modified electrode Cyclic voltammetry (CV) was used to form polymerization film. Prior to its modification, the bare GCE was polished with 0.05 m ␣-alumina powder and rinsed with 1:1 HNO3 solution, ethanol, and doubly distilled water for 10 min successively. After the electrode was pretreated electrochemically by scanning in a 0.5 mol L−1 H2 SO4 solution between −0.5 and 1.5 V at 100 mV s−1 for ten times to get a stable background current, the BCP modified electrode was prepared by electropolymerization. The polymertic film was deposited by cyclic sweeping from −1.0 to 1.5 V at 100 mV s−1 for 20 cycles in 0.01 mol L−1 NaNO3 solution containing 2.0 × 10−3 mol L−1 BCP. After polymerization, the modified electrode was washed with doubly distilled water, and then air-dried. 2.4. Experimental methods Cyclic voltammetric and differential pulse voltammetric measurements were carried out with three electrodes in phosphate buffer solution. The cyclic voltammograms were recorded by cycling the potential between 0.0 and +1.4 V at a scan rate of 100 mV s−1 . The differential pulse voltammetric measurements were performed by applying a sweep potential from 0.0 to +1.2 V at pulse amplitude of 50 mV and pulse width of 0.1 s. All experiments were carried out at room temperature. The poly (BCP) modified electrode could be used repeatedly after rinsed with doubly distilled water and blotted with filter paper. 2.5. Sample preparation Blood samples were collected from healthy volunteers at the Hospital of Shandong Normal University. A 1.0 mL of fresh blood sample was obtained and centrifuged at 3000 rpm for 20 min to remove all precipitating materials. After the separated serum was diluted 20-fold with pH 6.5 phosphate buffer solution, an aliquot 10.0 mL of this test solution was transferred into the electrochemical cell to detect UA, XA and HX simultaneously by the proposed DPV method.
2. Experimental
3. Results and discussion
2.1. Chemicals
3.1. Preparation and characterization of electropolymerized BCP film at the GCE surface
UA, XA and HX were purchased from Sigma (USA). BCP and ascorbic acid were obtained from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China). All chemicals were of analytical grade and were used without further purification. The 0.067 mol L−1 phosphate buffer solutions (PBS) with various pH values were prepared by mixing the stock solutions of 0.067 mol L−1 KH2 PO4 and Na2 HPO4 . All solutions were prepared with doubly distilled water.
2.2. Apparatus Electrochemical measurements were performed with a LK2005 Microcomputer-based electrochemical system (LANLIKE, Tianjin, China). A conventional three-electrode cell was used, including a saturated calomel electrode (SCE) as reference electrode, a platinum sheet electrode as the counter electrode and a bare or modified glassy carbon disk electrode (GCE) with a diameter of 3.0 mm used as working electrode. All pH measurements were made with a pHS-3C digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass electrode. A KQ250B ultrasonic washer (Kunshan Ultrasonic Instrument Works, Kunshan, China) was used to wash the electrode.
Cyclic voltammetry was used to form the electropolymerization film. The potential scan range was the most important factor in preparing poly (BCP) film. If the positive potential value for polymerization was below 1.0 V or if the negative one was above −0.8 V, no polymer reaction occurred. The experimental result showed that the polymeric film formed was more conductive when the potential scan window was from −1.0 to 1.5 V. Therefore, it was selected as the electropolymerization potential window in this paper. Compared with other supporting electrolyte such as phosphate buffer solution used in electrodeposition process of polymertic film, the obtained polymertic film was more complete, uniform and compact, which showed better electrocatalytic activity to the oxidation of UA, XA and HX when NaNO3 was used as supporting electrolyte during polymer formation. Thus NaNO3 was chosen as supporting electrolyte for electropolymerization in this work. The consecutive cyclic voltammograms of 2.0 × 10−3 mol L−1 BCP in 0.01 mol L−1 NaNO3 solution at bare glassy carbon electrode were shown in Fig. 1. In the first cycle, one strong anodic peak was observed at 0.800 V(peak A), which might correspond to the oxidation of BCP monomer. A cathodic peak also appeared at about −0.480 V (peak B) in the first cycle. From the second cycle on, a new oxidation peak appeared with potential at 0.520 V (peak C)
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Fig. 2. Cyclic voltammograms of poly (BCP) modified glassy carbon electrode in pH 6.5 phosphate buffer solution at different scan rates. (a) 10 mV s−1 ; (b) 20 mV s−1 ; (c) 40 mV s−1 ; (d) 60 mV s−1 ; (e) 80 mV s−1 ; (f) 100 mV s−1 . Fig. 1. Cyclic voltammograms of BCP in electropolymerization process from 1 to 20 cycles. BCP: 2.0 × 10−3 mol L−1 ; supporting electrolyte: 0.01 mol L−1 NaNO3 ; scan rate: 100 mV s−1 .
in the cyclic voltammograms, and its peak current increased with successive scanning, indicating that film formation has occurred. During the polymerized process, peak A and peak B descended gradually with cyclic time increasing. Both the anodic and cathodic peak currents tended to be stable after 18 scans. These facts suggest that the initially formed poly (BCP) film had a leaching process with scan cycle increasing up to 18 times, which maybe implied a self-adjustment of the polymer film thickness at the GCE [19]. So the total number of the electropolymerizing scans was selected as twenty for forming stable polymer film. After modification, a uniform adherent blue polymer was formed on the GCE surface, which demonstrated that BCP was deposited on the surface of GCE by electropolymerization. The electrochemical behavior of the poly (BCP) modified electrode was studied in phosphate buffer solution by CV. The cyclic voltammograms of the poly (BCP) film recorded in pH 6.5 phosphate buffer solution at different scan rates were shown in Fig. 2, in which a chemically reversible redox couple was obtained in each cycle. The anodic peak current (ipa ) was linearly dependent on the scan rate () over the range of 10–100 mV s−1 , with the regression equation ipa (A) = 0.5596 + 0.04106 (mV s−1 ) (correlation coefficient, r = 0.9983), indicating a surface-controlled electrode processes. Moreover, the ratio of anodic peak current to cathodic peak current (ipa /ipc ) was almost equal to unity. With increasing scan rate, the separation of peak potentials (Ep = Epa − Epc ) would not be changed. The above results suggest that the electrode reaction is reversible [20]. Ep (=58.3 mV) is
close to 2.3RT/nF (or 59/n mV at 25 ◦ C), which was on accordance with a Nernst reversible behavior. So the number of transferred electrons in the electrode reaction was identified as one (n ≈ 1.01). The surface coverage of poly (BCP) modified electrode was made by adopting the method used by Sharp et al. [21]. According to this method, the peak current is related to the surface concentration of electroactive species, by the following equation: ip =
n2 F 2 A 4RT
where n represents the number of electrons involved in reaction (n = 1), A is the surface geometrical area of electrode (0.0706 cm2 ), represents the surface coverage concentration (mol cm−2 ) and other symbols have their usual meaning. From the slope of anodic peak currents versus scan rate, the surface concentration of poly (BCP) was calculated as 6.81 × 10−8 mol cm−2 . The result in Fig. 2 proved that BCP had been deposited on the surface of GCE by electropolymerization, and the functional group in the poly (BCP) film had good redox activity. The effect of pH on the electrochemical behavior of the poly (BCP) modified electrode was studied in phosphate buffer solution over the pH range from 4.5 to 9.2. The results showed that both Epa and Epc shifted towards the positive direction with increasing pH, which indicated that protons have taken part in the electrode processes. The anodic peak potentials exhibited a linear dependence on pH values with a slope of 56.4 mV pH−1 , which suggested that the ratio of the participated protons to the transferred electrons through the poly (BCP) film is 1:1. Thus, the possible reaction mechanism of poly (BCP) film on the glassy carbon electrode can be expressed as follows (Scheme 1): BCP (A) was first deposited at
Scheme 1. Mechanism of the poly (BCP) electrode reaction.
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Fig. 3. Cyclic voltammograms of the mixture containing UA, XA and HX at the bare glassy carbon electrode (A) and the poly (BCP) modified electrode (B) in pH 6.5 phosphate buffer solution. UA: 4.0 × 10−5 mol L−1 , XA: 2.0 × 10−5 mol L−1 , HX: 2.0 × 10−5 mol L−1 and scan rate: 100 mV s−1 .
the surface of GCE and oxidized to form a benzoquinone structure (B), then it was subsequently reduced to BCP at the reverse scan. The modified electrode exhibited a high stability when it was placed in dry state. No loss of electroactivity of the electrode was found for the continuously cyclical sweeping for 6 h. The modified electrode was also not deteriorated even for one month. 3.2. Electrochemical behaviors of UA, XA and HX at poly (BCP) modified electrode Fig. 3 demonstrated the CVs of the mixture solution containing UA, XA and HX at bare GCE and modified electrode in pH 6.5 PBS. As shown in Fig. 3A, all three compounds exhibited poor current responds and no well-defined peak of their oxidation could be seen at bare GCE, indicating a slow electron transfer kinetic. So it is impossible to use the bare electrode for simultaneous determination of the three species. In great contrast, when the modified electrode was used, the mixture displayed three well-defined and sensitive oxidation peaks. The peaks located at 0.321, 0.707 and 1.062 V, corresponding to the oxidation of UA, XA and HX, respectively. The differences of the oxidation peak potentials for UA–XA and XA–HX were 386 and 355 mV, respectively, which were enough large separations to allow the simultaneous determination of UA, XA and HX in a mixture. Meanwhile, it could be noticed that the peak currents of UA, XA and HX were enhanced significantly and all the three oxidation peak potentials had negative potential shifts at the poly (BCP) modified electrode. The enhanced current response and the lowered overpotential clearly indicated that poly (BCP) film can accelerate the rate of electron transfer and have excellent electrocatalytic activity towards the oxidation of UA, XA and HX. The poly (BCP) film has high concentration of the negatively charged surface functional group SO3 − and of the electron-rich O atom, which could interact with the purine derivatives, such as UA, XA and HX. Moreover, the poly (BCP) modified electrode possessed the larger real surface area, – conjugated bond, a great deal of active sites and better conductivity, which led to the dissimilar conjugation effect of the purine derivatives with the electrode interface. Therefore, the electrochemical reversibility of the oxidation of UA, XA and HX may be greatly improved in the presence of the poly (BCP) film by accelerating the rate of electron transfer. UA, XA and HX could be identified entirely at the poly
Fig. 4. Cyclic voltammograms of UA (A), XA (B) and HX (C) at modified electrode at different scan rates. (a) 20 mV s−1 , (b) 40 mV s−1 , (c) 60 mV s−1 , (d) 80 mV s−1 , (e) 100 mV s−1 , (f) 150 mV s−1 , (g) 200 mV s−1 , (h) 250 mV s−1 ; UA: 4.0 × 10−5 mol L−1 , XA: 2.0 × 10−5 mol L−1 , HX: 2.0 × 10−5 mol L−1 ; pH = 6.5 PBS. The inset shows the plot of dependence of ipa on .
(BCP) modified electrode based on its high electrocatalytic activity. 3.3. Effect of scan rate on the oxidation of UA, XA and HX at poly (BCP) modified electrode Fig. 4A–C showed the CVs of UA, XA and HX recorded at poly (BCP) modified electrode at different potential scan rates (), respectively. The plots of ipa as a function of for three molecules were also shown in the insets of Fig. 4. For all the three compounds, the oxidation peak current linearly increased with the scan rate, suggesting that the system presents features corresponding to an adsorption-controlled process for UA, XA and HX [22]. The linear regression equation relating ipa with the
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the oxidation reaction of the three species. The equation relating Epa with pH was found to be: Epa (V) = 0.7281 − 0.0618 pH (r = 0.9986) for UA, Epa (V) = 1.069 − 0.0556 pH (r = 0.9995) for XA, and Epa (V) = 1.468 − 0.0625 pH (r = 0.9979) for HX, respectively. The slopes of 61.8 mV pH−1 for UA, 55.6 mV pH−1 for XA and 62.5 mV pH−1 for HX are close to the theoretical value of 59 mV pH−1 , suggesting that the electron transfer step is preceded by a protonation with an equal number of protons and electrons involved in their oxidation. The effect of pH on anodic peak current for UA, XA and HX was shown in Fig. 5A. It could be seen that the anodic peak current of UA decreased with increasing of pH in a range of 4.5–9.2. However, the anodic peak current for XA and HX increased with increasing the pH up to 6.5, then the peak current decreased. The maximum value was observed at pH 6.5 for both XA and HX. 3.5. Simultaneous determination of UA, XA and HX
Fig. 5. Effects of pH on the anodic peak current (A) and anodic peak potential (B) of UA, XA and HX at the modified electrode, respectively. UA: 1.0 × 10−4 mol L−1 , XA: 4.0 × 10−5 mol L−1 , HX: 4.0 × 10−5 mol L−1 ; scan rate: 100 mV s−1 ; phosphate buffer solution.
scan rate over the range of 20–250 mV s−1 , was found to be: ipa (A) = 5.922 + 0.1261 (mV s−1 ) for UA with a correlation coefficient (r) of 0.9985, ipa (A) = 10.13 + 0.2228 (mV s−1 ) (r = 0.9991) for XA, and ipa (A) = 7.696 + 0.1395 (mV s−1 ) (r = 0.9982) for HX, respectively. In addition, it was observed that the catalytic oxidation peak potential (Epa ) shifted to more positive potentials with increasing scan rate for all the species. The analysis of these data showed that the plot of Epa versus the logarithm of scan rate presented a linear relation, indicating that the electrocatalytic oxidation of UA, XA and HX on the modified electrode surface is irreversible [20]. 3.4. Effect of pH on the oxidation of UA, XA and HX at poly (BCP) modified electrode Fig. 5 illustrated the dependence of the anodic peak potential (Epa ) and anodic peak current (ipa ) for UA (1.0 × 10−4 mol L−1 ), XA (4.0 × 10−5 mol L−1 ) and HX (4.0 × 10−5 mol L−1 ) on pH of buffer solution. The peak potentials (Epa ) for UA, XA and HX showed a same trend and shift almost linearly towards negative potentials when pH was increased in the range of 4.5–9.2, indicating that protons are directly involved in the rate determination step of
According to the above experimental results, the modified electrode showed good electrocatalytic activity for oxidation reaction of UA, XA and HX in wide pH range. The maximum peak current value can be observed at pH 4.5 for UA and pH 6.5 for both XA and HX, respectively. Because pH 6.5 is closer than pH 4.5 to the physiological pH value and the oxidation of the three compounds had high electrochemical response at this pH, it was selected as the optimum pH for the simultaneous determination of UA, XA and HX in this study. Since differential pulse voltammetry (DPV) has higher sensitivity and better resolution than cyclic voltammetry, DPV was used for simultaneous determination of UA, XA and HX. The DPV parameters were selected as pulse amplitude of 50 mV and pulse width of 100 ms for quantitative determination of three purine derivatives in order to obtain maximum peak current. The results showed that the peak currents of DPV are linearly proportional to the concentrations of the three compounds, respectively. Under the optimum conditions, the linear ranges and detection limits of UA, XA and HX were shown in Table 1, respectively. After every determination, the modified electrode can be regenerated by cycling between 0.0 and 1.4 V in a blank solution for six times to increase its reproducibility. To test the repeatability of the modified electrode, 2.0 × 10−5 mol L−1 of the three species were measured for six times. The relative standard deviations (RSD) of the peak currents for UA, XA and HX were 1.6%, 1.1% and 2.3%, respectively. The same solutions were also determined with six electrodes made independently, and the interelectrode RSD for peak currents UA, XA and HX were 2.2%, 1.5% and 2.8%, respectively. The above results demonstrated that the proposed method had high sensitivity, wide linear range and good repeatability. The comparison of this method with other electrochemical methods for the simultaneous determination of UA, XA and HX was listed in Table 2. 3.6. Interferences In order to check the intermolecular effects between UA, XA and HX, three different experiments were carried out under optimum conditions. In each experiment, the concentration of one of the three compounds was changed, whereas the concentrations
Table 1 Analytical characteristics for simultaneous determination of UA, XA and HX by the proposed method. Analyte
Linear range (mol L−1 )
UA XA HX
5.0 × 10−7 –1.2 × 10−4 1.0 × 10−7 –1.0 × 10−4 2.0 × 10−7 –8.0 × 10−5
Linear regression equation (i: A, C: mol L−1 ) ipa = 1.983 + 0.1612C ipa = 2.072 + 0.6456C ipa = 1.491 + 0.3892C
Correlation coefficient
Detection limit (mol L−1 )
0.9991 0.9995 0.9986
2.0 × 10−7 6.0 × 10−8 1.2 × 10−7
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Table 2 Comparison of the proposed method with other electrochemical methods for the simultaneous determination of UA, XA and HX. Linear range (mol L−1 )
Electrode
−8
Detection limit (mol L−1 ) −4
Ref.
−8
Electrochemically pretreated carbon paste electrode
UA: 5.94 × 10 –1.19 × 10 XA: 1.31 × 10−7 –6.57 × 10−4 HX: 3.67 × 10−7 –7.35 × 10−5
UA: 2.97 × 10 XA: 6.57 × 10−8 HX: 7.35 × 10−8
[12]
Preanodized nontronite coated screen-printed carbon electrode
UA: 2.0 × 10−6 –4.0 × 10−5 XA: 2.0 × 10−6 –4.0 × 10−5 HX: 4.0 × 10−6 –3.0 × 10−5
UA: 4.2 × 10−7 XA: 7.0 × 10−8 HX: 3.4 × 10−7
[13]
Inlaying ultra-thin carbon paste electrode modified with functional single-wall carbon nanotubes
UA: 1.0 × 10−7 –1.0 × 10−4 XA: 2.0 × 10−7 –1.0 × 10−4 HX: 8.0 × 10−7 –1.0 × 10−4
UA: 8.0 × 10−8 XA: 1.46 × 10−7 HX: 5.62 × 10−7
[14]
Poly (ATD) modified glassy carbon electrode
UA: 5.0 × 10−6 –4.5 × 10−5 XA: 5.0 × 10−6 –4.5 × 10−5
UA: 1.9 × 10−7 XA: 5.9 × 10−7
[15]
Ru (DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode
UA: 1.0 × 10−4 –7.0 × 10−4 XA: 5.0 × 10−5 –5.0 × 10−4 HX: 5.0 × 10−5 –3.0 × 10−4
UA: 3.72 × 10−7 XA: 2.35 × 10−6 HX: 2.37 × 10−6
[16]
Poly (BCP) modified glassy carbon electrode
UA: 5.0 × 10−7 –1.2 × 10−4 XA: 1.0 × 10−7 –1.0 × 10−4 HX: 2.0 × 10−7 –8.0 × 10−5
UA: 2.0 × 10−7 XA: 6.0 × 10−8 HX: 1.2 × 10−7
This work
Table 3 Determination of UA, XA and HX in human serum samples (n = 6). Sample
1 2 3
Original (mol L−1 )
Added (mol L−1 )
Found (mol L−1 )
Recovery (%)
UA
XA
HX
UA
XA
HX
UA
XA
HX
UA
XA
HX
11.89 8.10 22.45
1.23 5.45 5.86
3.68 1.89 –
10.00 8.0 20.0
2.0 6.0 6.0
4.0 2.0 10.0
22.18 15.86 41.85
3.19 11.72 11.65
7.47 3.81 9.93
101.3 98.5 98.6
98.8 102.4 98.2
97.3 97.9 99.3
of the other two species remained constant. The results in Fig. 6 showed that no obvious changes in the peak currents (10-fold of UA can decrease the peak currents of XA and HX in the ratio of 1.8% and 1.2%, respectively) and potentials of both XA and HX could be found while varying the concentration of UA. In addition, the oxidation peak current of UA increased linearly with a correlation coefficient of 0.9991 while increasing UA concentrations. Similarly, the oxidation peak current of XA or HX increased with increasing its concentrations while keeping the concentrations of the other two compounds constant, no interference can be observed for the
determination of XA or HX by the coexisting other two species. All the results identified that the oxidation processes of UA, XA and HX at poly (BCP) modified electrode are independent from each other, so it is possible to simultaneously determine the three compounds using the modified electrode in real samples without any interference with each other. For evaluating selectivity of the modified electrode, various possible interfering species were examined for their effect on the determination of UA, XA and HX (all of them were 2.0 × 10−5 mol L−1 ). The tolerance limit was taken as the maximum concentration of the foreign substances that caused an approximately ±5% relative error in the determination. If the tested coexisting interferents brought the detection current signal deviation below 5% under sufferable coexisting amount, we considered that the above substances have no interference. The results showed that 100-fold of glucose, lactic acid, serine, 60-fold of cysteine, ascorbic acid, 20-fold of uracil, theophylline, 10-fold of caffeine, albumin and 5-fold of dopamine, adenine, guanine, did not interfere with the determination of UA, XA and HX. From the experiments of the interference, it seems that the proposed method can be applicable to the detection of real biological samples.
3.7. Analytical applications
Fig. 6. Differential pulse voltammograms for UA with different concentrations in the presence of 10.0 mol L−1 XA and 20.0 mol L−1 HX at the poly (BCP) modified electrode. UA concentrations: (a) 20.0 mol L−1 , (b) 40.0 mol L−1 , (c) 60.0 mol L−1 , (d) 80.0 mol L−1 , (e) 100.0 mol L−1 ; pulse amplitude: 50 mV; pulse width: 100ms; pH = 6.5 PBS.
The practical analytical utility of the modified electrode was illustrated by simultaneous determination of UA, XA and HX in human serum samples. After all the serum samples were diluted 20-fold with pH 6.5 phosphate buffer solution, the three compounds were determined simultaneously by the proposed DPV method. The obtained results were summarized in Table 3. It can be seen that all spike recoveries were accurate and precise, which indicated the good applicability of the poly (BCP) modified electrode to simultaneous determination of UA, XA and HX in the real samples.
Y. Wang, L.-l. Tong / Sensors and Actuators B 150 (2010) 43–49
4. Conclusions In this paper, the novel poly (BCP) modified electrode had been prepared by simple and fast electropolymerization method, and used as electrochemical sensor for the simultaneous determination of UA, XA and HX by DPV. The modified electrode had good stability, sensitivity and selectivity. The results demonstrated that the proposed method is a rapid, sensitive, and reproducible method for determination of purine derivatives in serum samples. Hence, we believe that the poly (BCP) modified electrode can become a useful tool for the assay of UA, XA and HX both in research assays and in clinical diagnosis due to its rapid speed, good precision and low cost of analysis. Acknowledgment The research presented in this manuscript was supported by Shandong Province Natural Science Foundation, China (No. Y2006B28). References [1] D.E. Metzler, Biochemistry: The Chemical Reactions of Living Cells, Academic Press, New York, 1977. [2] M. Heinig, R.J. Johnson, Role of uric acid in hypertension, renal disease, and metabolic syndrome, Clev. Clin. J. Med. 73 (2006) 1059–1064. [3] T. Yamamoto, Y. Moriwaki, S. Takahashi, Z. Tsutsumi, J. Yamakita, Y. Nasako, Determination of human plasma xanthine oxidase activity by highperformance liquid chromatography, J. Chromatogr. B: Biomed. Sci. Appl. 681 (1996) 395–400. [4] M. Czauderna, J. Kowalczyk, Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine by HPLC, J. Chromatogr. B: Biomed. Sci. Appl. 744 (2000) 129–138. [5] N. Cooper, R. Khosravan, C. Erdmann, J. Fiene, J.W. Lee, Quantification of uric acid, xanthine and hypoxanthine in human serum by HPLC for pharmacodynamic studies, J. Chromatogr. B: Biomed. Sci. Appl. 837 (2006) 1–10. [6] L. Terzuoli, B. Porcelli, C. Setacci, M. Guibbolini, Comparative determination of purine compounds in carotid plaque by capillary zone electrophoresis and high-performance liquid chromatography, J. Chromatogr. B: Biomed. Sci. Appl. 7289 (1999) 185–192. [7] M. Pizzichini, L. Arezzini, C. Billarelli, F. Carlucci, L. Terzuoli, Determination and separation of allantoin, uric acid, hypoxanthine, and xanthine by capillary zone electrophoresis, Adv. Exp. Med. Biol. 431 (1998) 797–800. [8] E. Caussˇıe, A. Pradelles, B. Dirat, A. Negre-Salvayre, R. Salvayre, F. Couderc, Simultaneous determination of allantoin, hypoxanthine, xanthine, and uric acid in serum/plasma by CE, Electrohporesis 28 (2007) 381–387. [9] M.A Carsol, G. Volpe, M. Mascini, Amperometric detection of uric acid and hypoxanthine with xanthine oxidase immobilized and carbon based screenprinted electrode: application for fish freshness determination, Talanta 44 (1997) 2151–2159.
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Biographies Yan Wang received her master degree in analytical chemistry in 1995 from Shandong University, PR China. She is currently an associate professor in the Department of Chemistry at Shandong Normal University, PR China. Her research interests include electroanalytical chemistry, bioanalytical chemistry, and capillary electrophoresis. Li-li Tong is a lecturer in the Department of Chemistry at Shandong Normal University, PR China. Her research area is bioanalytical chemistry.