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A highly sensitive electrochemical sensor for simultaneous detection of uric acid, xanthine and hypoxanthine based on poly(l -methionine) modified glassy carbon electrode
Sensors and Actuators B 188 (2013) 621–630
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A highly sensitive electrochemical sensor for simultaneous detection of uric acid, xanthine and hypoxanthine based on poly(l-methionine) modified glassy carbon electrode Reza Ojani ∗ , Ali Alinezhad, Zahra Abedi Electroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
a r t i c l e
i n f o
Article history: Received 12 March 2013 Received in revised form 24 May 2013 Accepted 8 July 2013 Available online xxx Keywords: Poly l-methionine Uric acid Xanthine Hypoxanthine Cyclic voltammetry Differential pulse voltammetry
a b s t r a c t A poly l-methionine modified GC electrode was fabricated by electrochemical polymerization of the l-methionine on a glassy carbon electrode. The electrochemical behaviors of uric acid, xanthine and hypoxanthine at the modified electrode were studied by cyclic voltammetry and differential pulse voltammetry. The results showed that this modified electrode exhibited excellent electrocatalytic activity toward the oxidation of the uric acid, xanthine and hypoxanthine. Also, electrode was used for simultaneous determination of uric acid, xanthine and hypoxanthine. This modified electrode possesses high sensitivity, and the detection limits as values 0.007, 0.004 and 0.008 M for uric acid, xanthine and hypoxanthine, respectively. Finally, the poly l-methionine modified electrode was successfully employed to detect uric acid, xanthine and hypoxanthine in the serum samples with good selectivity and high sensitivity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Uric acid (UA), xanthine (XA) and hypoxanthine (HX) are oxidation products of purine degradation metabolism in human body as shown in Scheme 1. Purine metabolite pathway involves transformation of HX → XA and XA → UA by xanthine oxidase, so XA and HX are intermediates and UA is the final product of purine degradation metabolism [1,2]. Abnormalities of the metabolite concentrations are sensitive indicators of certain pathologic states, including gout, xanthinuria, hyperuricemia, renal failure, toxemia during pregnancy, etc. [3,4]. Therefore, the measurement of UA, XA and HX is very important in the food, biochemical and clinical diagnosis [5]. Various techniques have been developed for simultaneously determination of the purine degradation products, such as enzymatic methods [6,7], high-performance liquid chromatography (HPLC) [8,9], capillary electrophoresis (CE) [10,11] and electrochemistry [6,12–14], in which HPLC methods require fastidious sample preparation, expensive material and prolonged analysis time, and CE methods need expensive apparatus. In contrast, electrochemical method has attracted significant attention as an alternative method because of its inherent advantages of simplicity, high sensitivity and relatively low cost [15]. However, only a few reports appeared for the simultaneous determination of UA,
∗ Corresponding author. Tel.: +98 112 5342301; fax: +98 112 5342302. E-mail address: [email protected] (R. Ojani). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.07.015
XA and HX such as poly (pyrocatechol violet)/functionalized multiwalled carbon nanotubes composite film modified electrode [15], Ru(DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode [5], the surface enhancement effect of mesoporous silica [16] and preanodized nontronite-coated screen-printed electrode [17]. Chemically modified electrodes (CMEs) have been largely used in the area of electrochemical and biological fields [18–26]. Amino acids are essential for lives and have been of interest to both chemists and biologists. Efforts have been made on their synthesis and determination [27–29]. However, few reports using amino acids as modifiers to decorate the electrodes have been reported in determining other compounds [30–32]. Therefore, in this work, we decided to prepare a new poly(l-methionine)/GCE modified electrode and to apply this electrode to catalyze the oxidation of UA, XA and HX and simultaneous determinations of them by voltammetric methods.
2. Experimental 2.1. Chemicals UA, XA, HX and l-methionine were purchased from Fluka. The 0.1 M phosphate buffer solutions (PBS) with various pH values were provided by mixing the stock solutions of 0.1 M NaH2 PO4 ·2H2 O, Na2 HPO4 ·2H2 O and 0.1 M KCl. All solutions are provided with distilled water.
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Fig. 1. Cyclic voltammograms of PLMT in electropolymerization process from 1 to 6 cycles PLMT: 0.01 M; supporting electrolyte: pH 7.20 phosphate buffer solution; scan rate: 100 mV s−1 . Scheme 1. Chemical structures of UA, XA and HX.
2.2. Instruments Electrochemical experiments were accomplished using a potentiostat/galvanostat (Bhp2061-C Electrochemical Analysis System, Behpajooh, Iran) and potentiostat/galvanostat (-Autolab TYPE III, Eco Chemie BV, Netherlands). A platinum wire was used as the counter electrode. A glassy carbon as working electrode and an SCE as reference electrode were used. A pH-meter (780 Metrohm) was used to read the pH of buffer solutions. 2.3. Preparation of the poly l-methionine/GCE The PLMT/GCE film modified electrode was prepared in the following way. Prior to its modification, the bare GCE was polished with 0.05 m alumina slurry and sonicated in ethanol and distilled water continuously. Then, the polymeric film was deposited by cycling the electrode potential between −0.6 V and 2 V at 100 mV s−1 for 6 cycles in PBS containing 2.5 × 10−3 M lmethionine at pH 7.20. The polymerization was studied at several different concentrations of methionine, but the concentration of 2.5 mM methionine has been shown to catalyze further. The effect of polymeric layer’s thickness was investigated by the sequence of different continuous cycle numbers during of the polymer preparation on the electrocatalytic ability of the polymer, that the six cycles of sweep potential were suitable. 2.4. Electrochemical methods Cyclic voltammetric and differential pulse voltammetric measurements were accomplished with three electrodes in phosphate
buffer solutions. The cyclic voltammograms were recorded by cycling the potential between 0.0 and +1.2 V at a scan rate of 100 mV s−1 . The differential pulse voltammetric measurements were carried out by applying a sweep potential from 0.0 to +1.2 V, at pulse amplitude of 50 mV, pulse width of 50 mV and scan rate 30 mV s−1 . All experiments were accomplished at room temperature. 2.5. Real sample preparation Blood samples were collected from healthy volunteers. 1.0 mL of fresh blood sample was taken and centrifuged at 4000 rpm for 10 min to separation of serum from blood samples. Afterwards the separated serum was attenuated 10-fold with pH 7.20 phosphate buffer solutions. Then, 10.0 ml of this test solution was transmigrated into the electrochemical cell to simultaneous determination XA, UA and HX by DPV method. 3. Results and discussion 3.1. Characterizations of poly l-methionine at the GCE surface Multi cyclic voltammetry of 2.5 × 10−3 mol l−1 l-methionine in 0.1 mol l−1 PBS solution at a GCE was studied (Fig. 1). In the first cycle, l-methionine showed an irreversible electrochemical oxidation. Its anodic peak appeared at potential of 1.40 V. During the following five cycles, this peak was diminished. This process was due to a thickening of polymer. The reaction mechanism may be described as follows [33]: l-Methionine was oxidized to free radical at the surface of the electrode,
The radicals then combine with the surface of GCE rapidly, and the product reacts as follows:
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Fig. 3. Nyquist plots of GC (a) and GC/PLMT (b) in the presence of 10 mM Fe3+ /Fe2+ in 0.1 M phosphate buffer solution pH 7.20.
equals to the electron transfer resistance, Rct . Fig. 3 shows the electrochemical impedance spectrograms of GCE and GCE/PLMT in the 10 mM Fe3+ /Fe2+ solution at potential 0.15 V, beginning frequency 10,000.0 Hz, end frequency 0.1 Hz, number of frequency 50 and amplitude (rms) 0.01 V On the bare GCE, the value of Rct was obtained 0.406 k (curve a). After immobilizing poly l-methionine at the GCE surface, the value of Rct was obtained 0.0803 k (curve b), which was smaller than that of bare GCE and was due to the presence of conductive poly l-methionine layer on the GCE. 3.4. Optimization of pH
Fig. 2. SEM images of (A) GCE and (B) GCE/PLMT.
3.2. SEM characterization Fig. 2 shows a typical morphology of GCE (A) and poly lmethionine/GCE (GCE/PLMT) (B). As can be seen in Fig. 2B, polymeric layer was established on the electrode surface. With the formation of polymeric layers on the GCE, the oxidation peak currents of UA, XA and HX were increased. This result can be attributed to the increasing of the real surface area. 3.3. Electrochemical impedance spectroscopy study Electrochemical impedance spectroscopy (EIS) is a valuable method to monitor the impedance changes of the electrode surface during the modification process [34]. Thus, it was used to monitor the assembly process. The semicircle part at higher frequencies, observed in the EIS, corresponds to the electron-transfer limited process and the linear part (after semi-circles) corresponds to diffusion process. The semicircle diameter in the impedance spectrum
The oxidation of UA, XA and HX is generally pH dependent [35]. Therefore, pH optimization of the solution seems necessary and was investigated to obtain the maximum sensitivity. The electrochemical behaviors of UA, XA and HX were studied at different pHs (in the pH range of 4.20–8.99) at the surface of the GC/PLMT by differential pulse voltammetry. Fig. 4A shows the effect of pH on cyclic voltammograms of HX. As you can see, the oxidation peak of HX has highest current at pH 7.20. The plot of the peak currents versus pH values for UA, XA and HX are shown in Fig. 4B. As can be seen, all three compounds have the highest current at pH = 7.20. Thus, pH = 7.20 was selected as the optimum pH for determination of these compounds. Also, Fig. 4C shows the variation of the peak potentials (Ep ) versus pH values for these compounds. Slope of curve Ep versus pH was obtained −0.0598 (±0.00090) V, −0.059 (±0.00081) V and −0.0581 (±0.0013) V for UA, XA and HX, respectively. The obtained slopes are close to the theoretical value of 0.059 V pH−1 and suggest that the same protons and electrons are involved in the oxidation process of these compounds. According to studies, the proposed mechanism for the three combinations is two electrons and two protons [36–38]. 3.5. Single oxidation of UA, XA and HX The electrochemical behavior of UA, XA and HX was investigated by cyclic voltammetry at the surface of the GCE and PLMT/GCE. Fig. 5 shows the cyclic voltammograms of UA, XA and HX at the surface of GCE and PLMT/GCE in 0.1 M PBS (pH 7.20) at scan rate of 100 mV s−1 . Curves (a) and (b) correspond to the GCE and PLMT/GCE in the presence of 50 M UA (A), 20 M XA (B) and 60 M HX (C), respectively. As can be seen in Fig. 5 the peak potentials due to the oxidation of UA, XA and HX occur in 0.27, 0.64 and 1.04 V at the surface of the bare GCE, with a peak currents of 2.8, 1.5 and 7 A, respectively. On
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investigated in a specific solution, containing UA, XA and HX, by using cyclic voltammetry. Fig. 6 shows the cyclic voltammograms of the solution containing 50 M UA, 20 M XA and 60 M HX at bare GCE and PLMT/GCE in PBS (pH 7.20). As can be seen in these voltammograms, three anodic peaks (I, II, III) appeared. Peaks I–III are related to the oxidation of UA, XA and HX, respectively. Therefore, we conclude that PLMT can perfectly distinguish between the electrooxidation of UA, XA and HX. 3.7. Effect of scan rate on the oxidation of UA, XA and HX at PLMT/GCE The kinetics of the electrode reactions were investigated by studying the effects of scan rate on the anodic peak currents of UA, XA and HX. Fig. 7 shows the CVs of 50 M UA solution on the PLMT/GCE at various scan rates. As shown in Fig. 7, the anodic peak currents of UA grows with the increasing the scan rates and there is a good linear relationship between the peak currents and scan rate (inset A) suggesting that the system presents features corresponding to an adsorption-controlled process for UA. Also, similar results were obtained for XA and HX. To obtain information about the rate-determining step, Tafel plots were drawn at scan rate of 20 mV s−1 for UA, XA and HX. Inset B in Fig. 7 shows a Tafel plot for UA. Based on Tafel plots, ˛ (transfer coefficient) values were obtained 0.30, 0.36 and 0.62 for UA, XA and HX, respectively. According to the Laviron theory, for an irreversible electrode process, Ep is calculated by the following equation [39]:
Ep = E 0 +
2.303RT ˛nF
log
RTk0 ˛nF
2.303RT +
˛nF
log
where ˛ is the transfer coefficient, k0 the standard heterogeneous rate constant for the reaction, n the number of electrons transferred, the scan rate of potential and E 0 is the formal redox potential. Other symbols have their usual meanings. The plot of Ep versus log is linear with an intercept that allows the k0 to be determined if the value of E 0 is known. The value of E 0 can be obtained from the intercept of the Ep versus curve by extrapolating to the vertical axis at = 0. For the oxidation of UA, XA and HX, E 0 were obtained 0 to be 0.246 V, 0.597 V and 0.910 V, and then the k was calculated to be 13.80 s−1 , 8.31 s−1 and 5.12 s−1 respectively.
Fig. 4. (A) Differential pulse voltammograms of a of 60 M HX in the pH range of 4.20–8.99. (B) Variation of the peak currents in the differential pulse voltammograms of 60 M HX (a), 0.1 mM UA (b) and 80 M XA (c). (C) Variation of the peak potentials in the differential pulse voltammograms of 60 M HX (a), 0.1 mM UA (b) and 80 M XA (c). Measurements carried out at the surface of the PLMT/GCE in 0.1 M buffer phosphate solution with various pH values, scan rates of potential is 30 mV s−1 .
the other hand, the oxidation potentials of UA, XA and HX at the surface of the PLMT/GCE decreased to 0.25, 0.62 and 0.95 V, with peak currents enhanced to 10.1, 17.8 and 32 A, respectively, compared with those at the GCE. This significant enhancement of the peak currents and shifting to less positive peak potentials at the surface of modified electrode confirms that PLMT facilitates and accelerates the kinetics of the electrochemical reactions and reduces the oxidation overpotentials of UA, XA and HX. 3.6. Simultaneous determination of HX, XA and UA at the PLMT/GCE For better establishment of the electrocatalytic effect of the PLMT at the surface of the electrode toward the oxidation of UA, XA and HX, the electrochemical behavior of these compounds was
3.8. Chronoamperometric studies Chronoamperometry was employed for investigating the electrocatalytic oxidation of UA, XA and HX. Fig. 8 depicts the current versus time curve of the modified electrode obtained by setting the working electrode potential at 0.64 V versus SCE for various concentrations of XA in PBS (pH 7.20). Also, for chronoamperometric study of UA and HX, working electrode potentials were set at 0.27 V and 0.97 V, respectively. Potential step chronoamperometry was used with poly l-methionine/GCE to determine the diffusion coefficient of UA, XA and HX. We have determined the diffusion coefficient, D, using the Cottrell’s equation [40]: I = nFAD1/2 Cb −1/2 t −1/2 where D and Cb , are the apparent diffusion coefficient (cm2 s−1 ) and the bulk concentration (mol cm−3 ), respectively. Inset A in Fig. 8 shows the plot of oxidation peak currents of cyclic voltammograms of XA versus t−1/2 at various concentrations. By using the slopes of the resulting straight lines plotted versus XA concentrations, the mean value of D = 8.34 × 10−5 cm2 s−1 obtained (inset B). Also, the mean values for UA and HX were found to be 8.71 × 10−5 cm2 s−1 and 1.66 × 10−4 cm2 s−1 respectively.
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Fig. 5. Cyclic voltammograms of 50 M UA (A), 20 M XA (B) and 60 M HX (C) in 0.1 M phosphate buffer solution pH 7.20 at the GCE (a) and PLMT/GCE (b) at a scan rate of 100 mV s−1 . Table 1 Analytical characteristics for simultaneous determination of UA, XA and HX at the PLMT/GC modified electrode. Analyte
Linear range(M)
Linear regression equation (i: A, C: M)
Correlation coefficient
Detection limit (M)
RSD (%)
LOQ (M)
RSD (%)
UA
0.02–0.1 0.1–1
Ipa = 19.518 (±0.010)C − 0.0089 (±0.0017) Ipa = 7.4374 (±0.022)C + 1.4042 (±0.038)
0.9962 0.9975
0.0074 0.05
1.2 0.8
0.021 0.15
1.5 1.3
XA
0.02–0.1 0.2–1
Ipa = 49.779 (±0.019)C + 0.0448 (±0.010) Ipa = 2.4642 (±0.026)C + 7.0085 (±0.011)
0.9984 0.9936
0.004 0.1
1.8 1.1
0.012 0.3
0.7 1.2
HX
0.02–0.1 0.2–1
Ipa = 25.752 (±0.025)C − 0.013 (±0.0050) Ipa = 2.45 (±0.029)C + 3.33 (±0.010)
0.9964 0.9921
0.008 0.097
0.6 1.3
0.024 0.29
1.6 0.9
Table 2 Comparison of the proposed method with other electrochemical methods for the simultaneous determination of UA, XA and HX. Electrode
Linear range (M)
Detection limit (M)
Ref.
Poly(l-arginine)/grapheme composite film modified electrode
UA: 0.1–10 XA: 0.1–10 HX: 0.2–20
UA: 0.05 XA: 0.05 HX: 0.10
[41]
Poly(pyrocatechol violet)/functional multi-walled carbon nanotubes composite film modified electrode
UA: 0.3–80 XA: 0.1–100 HX: 0.5–90
UA: 0.16 XA: 0.05 HX: 0.2
[16]
Poly(bromocresol purple) modified glassy carbon electrode
UA: 0.5–120 XA: 0.1–100 HX: 0.2–80
UA: 0.2 XA: 0.06 HX: 0.12
[35]
Ru(DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode
UA: 100–700 XA: 50–500 HX: 50–300
UA: 0.372 XA: 2.35 HX: 2.37
[5]
Preanodized nontronite coated screen-printed carbon electrode
UA: 2–40 XA: 2–40 HX: 4–30
UA: 0.42 XA: 0.07 HX: 0.32
[18]
Poly(l-methionine) modified glassy carbon electrode
UA: 0.02–0.1 XA: 0.02–0.1 HX: 0.02–0.1
UA: 0.0074 XA: 0.004 HX: 0.008
This work
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shows the DPV responses at the modified electrode while synchronously varying the concentration of HX. Also, all of the above studies were performed for UA and XA. The results showed that the peak currents of DPV are linearly proportional to the concentrations of these compounds. The linear ranges, detection limits and limits of quantification of UA, XA and HX are introduced in Table 1. The detection limit was obtained by using of the 3 (: blank standard deviation) divided by the slope of the calibration curve and LOQ was almost 3LOD. Linear range was obtained from the range between the lowest and highest observed concentration in the linear range of the calibration curve. The linear range and the detection limit for the simultaneous determination of UA, XA and HX at poly l-methionine/GCE were compared with the recently reported chemically modified electrodes in Table 2. According to Table 2, present work has lower LOD than that of other works. 3.10. Interference study
Fig. 6. Cyclic voltammograms of the phosphate buffer solution (pH 7.20) containing 50 M UA (I), 20 M XA (II) and 60 M HX (III) at bare GCE (a) and poly l-methionine/GCE (b) at scan rate 100 mV s−1 .
For evaluating selectivity of the poly(l-methionine)/GCE modified electrode, various possible interfering species were examined for their effects on the determination of UA, XA and HX (UA = 50 M, XA = 20 M and HX = 60 M). The results listed in Table 3. The recovery percent of UA, XA and HX were in the range 98.21–104.54%, which indicates that those species have no interference effects on the determinations of analytes. As you can see in Table 3, values of RSD (%) for UA, XA and HX were under 3%, indicating that the method has good accuracy.
3.9. Linear range, detection limits and limit of quantification 3.11. Reproducibility and stability of the modified electrode The Linear range, detection limits (LOD) and limit of quantification (LOQ) were individually studied using differential pulse voltammetry (DPV) in pH 7.20 phosphate buffer. Since DPV has a higher current sensitivity and better resolution than CV, it was used for simultaneous determination of UA, XA and HX. Fig. 9
In order to test the repeatability of the poly(l-methionine)/GCE modified electrode, the DPV for 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution were determined repeatedly at the identical surface of poly(l-methionine)/GCE. The intra-day
Fig. 7. Cyclic voltammograms of UA in 0.1 M phosphate buffer (pH 7.20), at various scan rates: a–g show 20, 40, 60, 80, 100, 150 and 200 mV s−1 , respectively. Insets: (A) variations of Ip versus scan rates and (B) Tafel plot at a scan rate of 20 mV s−1 .
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Fig. 8. Chronoamperograms obtained at PLMT/GCE in 0.1 M phosphate buffer solution (pH 7.20) for different concentrations of XA. The numbers 1 → 5 correspond to 0.0, 0.1, 0.5, 0.75 and 0.95 M. Insets: (A) plots of I versus t−1/2 obtained from chronoamperograms 2 → 5 and (B) plot of the slope of the straight lines against the XA concentration.
Fig. 9. Differential pulse voltammograms of a fixed concentration of UA (1.0 mM) and XA (1.0 mM) and different concentrations of HX: (a) 0.02, (b) 0.04, (c) 0.06, (d) 0.1, (e) 0.2, (f) 0.4, (g) 0.6, (h) 0.8 and (i) 1 M in 0.1 M phosphate buffer solution (pH 7.20) at the PLMT/GCE. Inset: analytical plot for HX voltammetric determination obtained from its DPV data. (In all the calibration plots, each data point is the average of three current values.)
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Table 3 Effect of different interferents on the voltammetric signal of UA, XA and HX at the PLMT/GCE. Interfering agent
Dopamine Ascorbic acid Epinephrine
Interfering agent
80 1000 100
Recovery (%)
RSD (%)
UA
XA
HX
UA
XA
HX
100.00 104.54 100.00
102.22 98.21 99.23
100.00 103.33 98.87
1.5 2.1 1.7
1.1 1.6 0.8
2.7 1.3 1.5
Fig. 11. Differential pulse voltammograms of UA, XA and HX in human serum samples (a) blank serum sample was attenuated 10-fold with pH 7.20 phosphate buffer solutions; (b) serum sample spiked with 0.02 M UA, XA and HX at scan rate 30 mV s−1 .
Fig. 10. Differential pulse voltammograms of UA (50 M), XA (30 M) and HX (40 M) in 0.1 M phosphate buffer solution with pH 7.20 at the PLMT/GCE, (a) one day, (b) after a week and (c) after ten days at a scan rate of 30 mV s−1 .
the laboratory, the modified electrode retains 97.56%, 98.08% and 99.60% for UA, XA and HX, respectively of its initial response after a week and 91.46%, 90.38% and 92.40% after 10 days. The above results demonstrated that the proposed method had good repeatability, excellent precision and long-term stability. 3.12. Analytical applications
precision of the method was evaluated by repeating five experiments in the same solution containing 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution taken separately using the same poly(l-methionine)/GCE modified electrode. The RSD was found to be 1.25%, 1.92% and 1.6% for UA, XA and HX, respectively, indicating excellent reproducibility of the modified electrode. Further, inter-day precision was investigated by measuring the current response of the modified electrode for 7 consecutive days for the same concentration of 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution taken separately and the respective relative standard deviations were found to be 1.81%, 1.12% and 1.74%. Thus, it demonstrated the good reproducibility of the method using the poly(l-methionine)/GCE modified electrode. The same solutions were also determined with 5 modified electrodes made independently, and the RSD for peak currents of UA, XA and HX were 1.85%, 1.47% and 1.85%, respectively. In order to test the stability of the poly(l-methionine)/GCE modified electrode, the DPVs for 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution were determined repeatedly at the identical surface of poly(l-methionine)/GCE (Fig. 10). When the electrode is stored in
The analytical utility of the modified electrode was illustrated by simultaneous determination of UA, XA and HX in human serum samples. The serum samples were diluted 10-fold with pH 7.20 phosphate buffer solution and the three compounds were determined simultaneously by the DPV method. Curves a and b in Fig. 11 represent differential pulse voltammograms for blank serum sample and a spiked serum sample containing standard solution of UA (2.0 × 10−8 M), XA (2.0 × 10−8 M) and HX (2.0 × 10−8 M), respectively. The DPV of serum sample (Fig. 11a) showed three oxidation peaks at 0.23 V, 0.59 V and 0.95 V, respectively. To confirm the observed oxidation peaks are due to the oxidation of UA, XA and HX, the serum sample was spiked with known concentrations of UA, XA and HX. The current response at 0.24 V, 0.59 V and 0.95 V enhanced after the addition of UA, XA and HX (Fig. 11b), which demonstrated that the observed oxidation peaks are due to the oxidation of the three compounds. For the recovery, all the serum samples were diluted 100-fold with pH 7.20 phosphate buffer solutions. The concentrations of UA, XA and HX were determined using the standard addition method, and the results are listed in Table 4.
Table 4 Simultaneous determination of UA, XA and HX in serum sample. No. Exp.
1 2 3
UA spiking, 10−7 M
XA spiking, 10−7 M
HX spiking, 10−7 M
UA found, 10−7 M
XA found, 10−7 M
HX found, 10−7 M
0.5 0.6 0.5
0.3 0.4 0.3
0.2 0.5 0.2
0.51 0.58 0.52
0.32 0.41 0.30
0.22 0.50 0.20
Recovery (%)
RSD (%)
UA
XA
HX
UA
XA
HX
101.4 97.3 104.0
106.7 102.7 100.9
109.0 99.4 99.0
1.7 0.9 1.9
1.8 1.5 2.1
1.2 1.6 2.4
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It can be seen that all spike recoveries were accurate and precise, which indicated that the proposed method could be efficiently used for simultaneous detection of UA, XA and HX in real samples. 4. Conclusions In this work, the poly(l-methionine) modified electrode prepared by simple and fast electropolymerization method and we applied this electrode for the simultaneous determination of UA, XA and HX by cyclic voltammetry, chronoamperometry and differential pulse voltammetry. The results showed that the oxidation of UA, XA and HX is catalyzed at pH 7.20, where the peak potential of these compounds is shifted to less positive peak potentials at the surface of the PLMT. This electrode exhibits high selectivity, excellent sensitivity, good reproducibility, stability and accuracy in the voltammetric measurement of UA, XA and HX. Also, the modified electrode can be applied to the determination of purine derivatives in human serum samples with satisfactory results. References [1] H. John, T. Ayvazian, S. Skupp, The study of purine utilization and excretion in a xanthinuric man, Journal of Clinical Investigation 44 (1965) 1248–1260. [2] T. Yamamoto, Y. Moriwaki, S. Takahashi, Effect of ethanol on metabolism of purine bases (hypoxanthine, xanthine, and uric acid), Clinica Chimica Acta 356 (2005) 35–57. [3] N.V. Bhagavan, Medical Biochemistry, fourth ed., Academic Press, USA, 2002. [4] J.S.N. Dutt, M.F. Cardosi, J. Davis, Electrochemical tagging of urate: developing new redox probes, Analyst 128 (2003) 811–813. [5] 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, Journal of Electroanalytical Chemistry 642 (2010) 135–142. [6] 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. [7] E.E. Kelley, A. Trostchansky, H. Rubbo, B.A. Freeman, R. Radi, M.M. Tarpey, Binding of xanthine oxidase to glycosaminoglycans limits inhibition by oxypurinol, Journal of Biological Chemistry 279 (2004) 37231–37234. [8] M. Czauderna, J. Kowalczyk, Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine urine by high-performance liquid chromatography and photodiode array detection, Journal of Chromatography B: Biomedical Sciences and Applications 744 (2000) 129–138. [9] 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, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 837 (2006) 1–10. [10] F. Carlucci, A. Tabucchi, B. Biagioli, G. Sani, G. Lisi, M. Maccherini, F. Rosi, E. Marinello, Capillary electrophoresis in the evaluation of ischemic injury: simultaneous determination of purine compounds and glutathione, Electrophoresis 21 (2000) 1552–1557. [11] E. Caussé, 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, Electrophoresis 28 (2007) 381–387. [12] A.M. Cubukcu, S. Timur, U. Anik, Examination of the performance of the glassy carbon paste electrode modified with gold nanoparticle and xanthine oxidase for xanthine and hypoxanthine detection, Talanta 74 (2007) 434–439. [13] G. Park, R.N. Adams, W.R. White, A rapid accurate electrochemical method for serum uric acid, Analytical Letters 5 (1972) 887–896. [14] R.N. Goyal, A. Mittal, S. Sharma, Simultaneous voltammetric determination of hypoxanthine, xanthine and uric acid, Electroanalysis 6 (1994) 609–611. [15] Y. Wang, Simultaneous determination of uric acid, xanthine and hypoxanthine at poly(pyrocatechol violet)/functionalized multi-walled carbon nanotubes composite film modified electrode, Colloids and Surfaces B: Biointerfaces 88 (2011) 614–621. [16] D. Sun, Y. Zhang, F. Wang, K. Wu, J. Chen, Y. Zhou, Electrochemical sensor for simultaneous detection of ascorbic acid, uric acid and xanthine based on the surface enhancement effect of mesoporous silica, Sensors and Actuators B: Chemical 14 (2009) 641–645. [17] J.M. Zen, Y.Y. Lai, H.H. Yang, A.S. Kumar, Multianalyte sensor for the simultaneous determination of hypoxanthine, xanthine and uric acid based on a preanodized nontronite-coated screen-printed electrode, Sensors and Actuators B: Chemical 84 (2002) 237–244. [18] C.W. Xu, R. Zeng, P.K. Shen, Z.D. Wei, Synergistic effect of CeO2 modified Pt/C catalysts on the alcohols oxidation, Electrochimica Acta 51 (2005) 1031–1035. [19] T. Inoue, J.R. Kirchhoff, Electrochemical detection of thiols with a coenzyme pyrrologuinoline quinone modified electrode, Analytical Chemistry 72 (2000) 5755–5760.
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Biographies Reza Ojani is a full professor of analytical chemistry, Mazandaran University, Babolsar, Iran. He received BSc from Gilan University, 1988; MSc from Tabriz University in analytical chemistry, 1991; and PhD from Tabriz University in analytical chemistry, 1996. His research interests cover electrochemical sensors, biosensors, electrochemical behavior of nanoparticles and fuel cell.
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Ali Alinezhad received the BSc degree in applied chemistry from Tabriz University, Iran, in 1996; MSc from Mazandaran University in analytical chemistry, 1999; and PhD from Mazandaran University in analytical chemistry, 2008. His research interests electrocatalyze, modified electrodes, electrosynthesis and electrochemistry in aqueous environments micelle.
Zahra Abedi received the BSc degree in applied chemistry from Payam Noor University, Behshar, Iran, in 2008. Now, she is working toward the MSc degree at Mazandaran University, Babolsar, Iran. Her interests are analytical chemistry, bioelectroanalytical chemistry and electrochemical properties of nanomaterials.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
A highly sensitive electrochemical sensor for simultaneous detection of uric acid, xanthine and hypoxanthine based on poly(l-methionine) modified glassy carbon electrode Reza Ojani ∗ , Ali Alinezhad, Zahra Abedi Electroanalytical Chemistry Research Laboratory, Department of Analytical Chemistry, Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
a r t i c l e
i n f o
Article history: Received 12 March 2013 Received in revised form 24 May 2013 Accepted 8 July 2013 Available online xxx Keywords: Poly l-methionine Uric acid Xanthine Hypoxanthine Cyclic voltammetry Differential pulse voltammetry
a b s t r a c t A poly l-methionine modified GC electrode was fabricated by electrochemical polymerization of the l-methionine on a glassy carbon electrode. The electrochemical behaviors of uric acid, xanthine and hypoxanthine at the modified electrode were studied by cyclic voltammetry and differential pulse voltammetry. The results showed that this modified electrode exhibited excellent electrocatalytic activity toward the oxidation of the uric acid, xanthine and hypoxanthine. Also, electrode was used for simultaneous determination of uric acid, xanthine and hypoxanthine. This modified electrode possesses high sensitivity, and the detection limits as values 0.007, 0.004 and 0.008 M for uric acid, xanthine and hypoxanthine, respectively. Finally, the poly l-methionine modified electrode was successfully employed to detect uric acid, xanthine and hypoxanthine in the serum samples with good selectivity and high sensitivity. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Uric acid (UA), xanthine (XA) and hypoxanthine (HX) are oxidation products of purine degradation metabolism in human body as shown in Scheme 1. Purine metabolite pathway involves transformation of HX → XA and XA → UA by xanthine oxidase, so XA and HX are intermediates and UA is the final product of purine degradation metabolism [1,2]. Abnormalities of the metabolite concentrations are sensitive indicators of certain pathologic states, including gout, xanthinuria, hyperuricemia, renal failure, toxemia during pregnancy, etc. [3,4]. Therefore, the measurement of UA, XA and HX is very important in the food, biochemical and clinical diagnosis [5]. Various techniques have been developed for simultaneously determination of the purine degradation products, such as enzymatic methods [6,7], high-performance liquid chromatography (HPLC) [8,9], capillary electrophoresis (CE) [10,11] and electrochemistry [6,12–14], in which HPLC methods require fastidious sample preparation, expensive material and prolonged analysis time, and CE methods need expensive apparatus. In contrast, electrochemical method has attracted significant attention as an alternative method because of its inherent advantages of simplicity, high sensitivity and relatively low cost [15]. However, only a few reports appeared for the simultaneous determination of UA,
∗ Corresponding author. Tel.: +98 112 5342301; fax: +98 112 5342302. E-mail address: [email protected] (R. Ojani). 0925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.snb.2013.07.015
XA and HX such as poly (pyrocatechol violet)/functionalized multiwalled carbon nanotubes composite film modified electrode [15], Ru(DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode [5], the surface enhancement effect of mesoporous silica [16] and preanodized nontronite-coated screen-printed electrode [17]. Chemically modified electrodes (CMEs) have been largely used in the area of electrochemical and biological fields [18–26]. Amino acids are essential for lives and have been of interest to both chemists and biologists. Efforts have been made on their synthesis and determination [27–29]. However, few reports using amino acids as modifiers to decorate the electrodes have been reported in determining other compounds [30–32]. Therefore, in this work, we decided to prepare a new poly(l-methionine)/GCE modified electrode and to apply this electrode to catalyze the oxidation of UA, XA and HX and simultaneous determinations of them by voltammetric methods.
2. Experimental 2.1. Chemicals UA, XA, HX and l-methionine were purchased from Fluka. The 0.1 M phosphate buffer solutions (PBS) with various pH values were provided by mixing the stock solutions of 0.1 M NaH2 PO4 ·2H2 O, Na2 HPO4 ·2H2 O and 0.1 M KCl. All solutions are provided with distilled water.
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Fig. 1. Cyclic voltammograms of PLMT in electropolymerization process from 1 to 6 cycles PLMT: 0.01 M; supporting electrolyte: pH 7.20 phosphate buffer solution; scan rate: 100 mV s−1 . Scheme 1. Chemical structures of UA, XA and HX.
2.2. Instruments Electrochemical experiments were accomplished using a potentiostat/galvanostat (Bhp2061-C Electrochemical Analysis System, Behpajooh, Iran) and potentiostat/galvanostat (-Autolab TYPE III, Eco Chemie BV, Netherlands). A platinum wire was used as the counter electrode. A glassy carbon as working electrode and an SCE as reference electrode were used. A pH-meter (780 Metrohm) was used to read the pH of buffer solutions. 2.3. Preparation of the poly l-methionine/GCE The PLMT/GCE film modified electrode was prepared in the following way. Prior to its modification, the bare GCE was polished with 0.05 m alumina slurry and sonicated in ethanol and distilled water continuously. Then, the polymeric film was deposited by cycling the electrode potential between −0.6 V and 2 V at 100 mV s−1 for 6 cycles in PBS containing 2.5 × 10−3 M lmethionine at pH 7.20. The polymerization was studied at several different concentrations of methionine, but the concentration of 2.5 mM methionine has been shown to catalyze further. The effect of polymeric layer’s thickness was investigated by the sequence of different continuous cycle numbers during of the polymer preparation on the electrocatalytic ability of the polymer, that the six cycles of sweep potential were suitable. 2.4. Electrochemical methods Cyclic voltammetric and differential pulse voltammetric measurements were accomplished with three electrodes in phosphate
buffer solutions. The cyclic voltammograms were recorded by cycling the potential between 0.0 and +1.2 V at a scan rate of 100 mV s−1 . The differential pulse voltammetric measurements were carried out by applying a sweep potential from 0.0 to +1.2 V, at pulse amplitude of 50 mV, pulse width of 50 mV and scan rate 30 mV s−1 . All experiments were accomplished at room temperature. 2.5. Real sample preparation Blood samples were collected from healthy volunteers. 1.0 mL of fresh blood sample was taken and centrifuged at 4000 rpm for 10 min to separation of serum from blood samples. Afterwards the separated serum was attenuated 10-fold with pH 7.20 phosphate buffer solutions. Then, 10.0 ml of this test solution was transmigrated into the electrochemical cell to simultaneous determination XA, UA and HX by DPV method. 3. Results and discussion 3.1. Characterizations of poly l-methionine at the GCE surface Multi cyclic voltammetry of 2.5 × 10−3 mol l−1 l-methionine in 0.1 mol l−1 PBS solution at a GCE was studied (Fig. 1). In the first cycle, l-methionine showed an irreversible electrochemical oxidation. Its anodic peak appeared at potential of 1.40 V. During the following five cycles, this peak was diminished. This process was due to a thickening of polymer. The reaction mechanism may be described as follows [33]: l-Methionine was oxidized to free radical at the surface of the electrode,
The radicals then combine with the surface of GCE rapidly, and the product reacts as follows:
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Fig. 3. Nyquist plots of GC (a) and GC/PLMT (b) in the presence of 10 mM Fe3+ /Fe2+ in 0.1 M phosphate buffer solution pH 7.20.
equals to the electron transfer resistance, Rct . Fig. 3 shows the electrochemical impedance spectrograms of GCE and GCE/PLMT in the 10 mM Fe3+ /Fe2+ solution at potential 0.15 V, beginning frequency 10,000.0 Hz, end frequency 0.1 Hz, number of frequency 50 and amplitude (rms) 0.01 V On the bare GCE, the value of Rct was obtained 0.406 k (curve a). After immobilizing poly l-methionine at the GCE surface, the value of Rct was obtained 0.0803 k (curve b), which was smaller than that of bare GCE and was due to the presence of conductive poly l-methionine layer on the GCE. 3.4. Optimization of pH
Fig. 2. SEM images of (A) GCE and (B) GCE/PLMT.
3.2. SEM characterization Fig. 2 shows a typical morphology of GCE (A) and poly lmethionine/GCE (GCE/PLMT) (B). As can be seen in Fig. 2B, polymeric layer was established on the electrode surface. With the formation of polymeric layers on the GCE, the oxidation peak currents of UA, XA and HX were increased. This result can be attributed to the increasing of the real surface area. 3.3. Electrochemical impedance spectroscopy study Electrochemical impedance spectroscopy (EIS) is a valuable method to monitor the impedance changes of the electrode surface during the modification process [34]. Thus, it was used to monitor the assembly process. The semicircle part at higher frequencies, observed in the EIS, corresponds to the electron-transfer limited process and the linear part (after semi-circles) corresponds to diffusion process. The semicircle diameter in the impedance spectrum
The oxidation of UA, XA and HX is generally pH dependent [35]. Therefore, pH optimization of the solution seems necessary and was investigated to obtain the maximum sensitivity. The electrochemical behaviors of UA, XA and HX were studied at different pHs (in the pH range of 4.20–8.99) at the surface of the GC/PLMT by differential pulse voltammetry. Fig. 4A shows the effect of pH on cyclic voltammograms of HX. As you can see, the oxidation peak of HX has highest current at pH 7.20. The plot of the peak currents versus pH values for UA, XA and HX are shown in Fig. 4B. As can be seen, all three compounds have the highest current at pH = 7.20. Thus, pH = 7.20 was selected as the optimum pH for determination of these compounds. Also, Fig. 4C shows the variation of the peak potentials (Ep ) versus pH values for these compounds. Slope of curve Ep versus pH was obtained −0.0598 (±0.00090) V, −0.059 (±0.00081) V and −0.0581 (±0.0013) V for UA, XA and HX, respectively. The obtained slopes are close to the theoretical value of 0.059 V pH−1 and suggest that the same protons and electrons are involved in the oxidation process of these compounds. According to studies, the proposed mechanism for the three combinations is two electrons and two protons [36–38]. 3.5. Single oxidation of UA, XA and HX The electrochemical behavior of UA, XA and HX was investigated by cyclic voltammetry at the surface of the GCE and PLMT/GCE. Fig. 5 shows the cyclic voltammograms of UA, XA and HX at the surface of GCE and PLMT/GCE in 0.1 M PBS (pH 7.20) at scan rate of 100 mV s−1 . Curves (a) and (b) correspond to the GCE and PLMT/GCE in the presence of 50 M UA (A), 20 M XA (B) and 60 M HX (C), respectively. As can be seen in Fig. 5 the peak potentials due to the oxidation of UA, XA and HX occur in 0.27, 0.64 and 1.04 V at the surface of the bare GCE, with a peak currents of 2.8, 1.5 and 7 A, respectively. On
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investigated in a specific solution, containing UA, XA and HX, by using cyclic voltammetry. Fig. 6 shows the cyclic voltammograms of the solution containing 50 M UA, 20 M XA and 60 M HX at bare GCE and PLMT/GCE in PBS (pH 7.20). As can be seen in these voltammograms, three anodic peaks (I, II, III) appeared. Peaks I–III are related to the oxidation of UA, XA and HX, respectively. Therefore, we conclude that PLMT can perfectly distinguish between the electrooxidation of UA, XA and HX. 3.7. Effect of scan rate on the oxidation of UA, XA and HX at PLMT/GCE The kinetics of the electrode reactions were investigated by studying the effects of scan rate on the anodic peak currents of UA, XA and HX. Fig. 7 shows the CVs of 50 M UA solution on the PLMT/GCE at various scan rates. As shown in Fig. 7, the anodic peak currents of UA grows with the increasing the scan rates and there is a good linear relationship between the peak currents and scan rate (inset A) suggesting that the system presents features corresponding to an adsorption-controlled process for UA. Also, similar results were obtained for XA and HX. To obtain information about the rate-determining step, Tafel plots were drawn at scan rate of 20 mV s−1 for UA, XA and HX. Inset B in Fig. 7 shows a Tafel plot for UA. Based on Tafel plots, ˛ (transfer coefficient) values were obtained 0.30, 0.36 and 0.62 for UA, XA and HX, respectively. According to the Laviron theory, for an irreversible electrode process, Ep is calculated by the following equation [39]:
Ep = E 0 +
2.303RT ˛nF
log
RTk0 ˛nF
2.303RT +
˛nF
log
where ˛ is the transfer coefficient, k0 the standard heterogeneous rate constant for the reaction, n the number of electrons transferred, the scan rate of potential and E 0 is the formal redox potential. Other symbols have their usual meanings. The plot of Ep versus log is linear with an intercept that allows the k0 to be determined if the value of E 0 is known. The value of E 0 can be obtained from the intercept of the Ep versus curve by extrapolating to the vertical axis at = 0. For the oxidation of UA, XA and HX, E 0 were obtained 0 to be 0.246 V, 0.597 V and 0.910 V, and then the k was calculated to be 13.80 s−1 , 8.31 s−1 and 5.12 s−1 respectively.
Fig. 4. (A) Differential pulse voltammograms of a of 60 M HX in the pH range of 4.20–8.99. (B) Variation of the peak currents in the differential pulse voltammograms of 60 M HX (a), 0.1 mM UA (b) and 80 M XA (c). (C) Variation of the peak potentials in the differential pulse voltammograms of 60 M HX (a), 0.1 mM UA (b) and 80 M XA (c). Measurements carried out at the surface of the PLMT/GCE in 0.1 M buffer phosphate solution with various pH values, scan rates of potential is 30 mV s−1 .
the other hand, the oxidation potentials of UA, XA and HX at the surface of the PLMT/GCE decreased to 0.25, 0.62 and 0.95 V, with peak currents enhanced to 10.1, 17.8 and 32 A, respectively, compared with those at the GCE. This significant enhancement of the peak currents and shifting to less positive peak potentials at the surface of modified electrode confirms that PLMT facilitates and accelerates the kinetics of the electrochemical reactions and reduces the oxidation overpotentials of UA, XA and HX. 3.6. Simultaneous determination of HX, XA and UA at the PLMT/GCE For better establishment of the electrocatalytic effect of the PLMT at the surface of the electrode toward the oxidation of UA, XA and HX, the electrochemical behavior of these compounds was
3.8. Chronoamperometric studies Chronoamperometry was employed for investigating the electrocatalytic oxidation of UA, XA and HX. Fig. 8 depicts the current versus time curve of the modified electrode obtained by setting the working electrode potential at 0.64 V versus SCE for various concentrations of XA in PBS (pH 7.20). Also, for chronoamperometric study of UA and HX, working electrode potentials were set at 0.27 V and 0.97 V, respectively. Potential step chronoamperometry was used with poly l-methionine/GCE to determine the diffusion coefficient of UA, XA and HX. We have determined the diffusion coefficient, D, using the Cottrell’s equation [40]: I = nFAD1/2 Cb −1/2 t −1/2 where D and Cb , are the apparent diffusion coefficient (cm2 s−1 ) and the bulk concentration (mol cm−3 ), respectively. Inset A in Fig. 8 shows the plot of oxidation peak currents of cyclic voltammograms of XA versus t−1/2 at various concentrations. By using the slopes of the resulting straight lines plotted versus XA concentrations, the mean value of D = 8.34 × 10−5 cm2 s−1 obtained (inset B). Also, the mean values for UA and HX were found to be 8.71 × 10−5 cm2 s−1 and 1.66 × 10−4 cm2 s−1 respectively.
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Fig. 5. Cyclic voltammograms of 50 M UA (A), 20 M XA (B) and 60 M HX (C) in 0.1 M phosphate buffer solution pH 7.20 at the GCE (a) and PLMT/GCE (b) at a scan rate of 100 mV s−1 . Table 1 Analytical characteristics for simultaneous determination of UA, XA and HX at the PLMT/GC modified electrode. Analyte
Linear range(M)
Linear regression equation (i: A, C: M)
Correlation coefficient
Detection limit (M)
RSD (%)
LOQ (M)
RSD (%)
UA
0.02–0.1 0.1–1
Ipa = 19.518 (±0.010)C − 0.0089 (±0.0017) Ipa = 7.4374 (±0.022)C + 1.4042 (±0.038)
0.9962 0.9975
0.0074 0.05
1.2 0.8
0.021 0.15
1.5 1.3
XA
0.02–0.1 0.2–1
Ipa = 49.779 (±0.019)C + 0.0448 (±0.010) Ipa = 2.4642 (±0.026)C + 7.0085 (±0.011)
0.9984 0.9936
0.004 0.1
1.8 1.1
0.012 0.3
0.7 1.2
HX
0.02–0.1 0.2–1
Ipa = 25.752 (±0.025)C − 0.013 (±0.0050) Ipa = 2.45 (±0.029)C + 3.33 (±0.010)
0.9964 0.9921
0.008 0.097
0.6 1.3
0.024 0.29
1.6 0.9
Table 2 Comparison of the proposed method with other electrochemical methods for the simultaneous determination of UA, XA and HX. Electrode
Linear range (M)
Detection limit (M)
Ref.
Poly(l-arginine)/grapheme composite film modified electrode
UA: 0.1–10 XA: 0.1–10 HX: 0.2–20
UA: 0.05 XA: 0.05 HX: 0.10
[41]
Poly(pyrocatechol violet)/functional multi-walled carbon nanotubes composite film modified electrode
UA: 0.3–80 XA: 0.1–100 HX: 0.5–90
UA: 0.16 XA: 0.05 HX: 0.2
[16]
Poly(bromocresol purple) modified glassy carbon electrode
UA: 0.5–120 XA: 0.1–100 HX: 0.2–80
UA: 0.2 XA: 0.06 HX: 0.12
[35]
Ru(DMSO)4 Cl2 nano-aggregated Nafion membrane modified electrode
UA: 100–700 XA: 50–500 HX: 50–300
UA: 0.372 XA: 2.35 HX: 2.37
[5]
Preanodized nontronite coated screen-printed carbon electrode
UA: 2–40 XA: 2–40 HX: 4–30
UA: 0.42 XA: 0.07 HX: 0.32
[18]
Poly(l-methionine) modified glassy carbon electrode
UA: 0.02–0.1 XA: 0.02–0.1 HX: 0.02–0.1
UA: 0.0074 XA: 0.004 HX: 0.008
This work
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shows the DPV responses at the modified electrode while synchronously varying the concentration of HX. Also, all of the above studies were performed for UA and XA. The results showed that the peak currents of DPV are linearly proportional to the concentrations of these compounds. The linear ranges, detection limits and limits of quantification of UA, XA and HX are introduced in Table 1. The detection limit was obtained by using of the 3 (: blank standard deviation) divided by the slope of the calibration curve and LOQ was almost 3LOD. Linear range was obtained from the range between the lowest and highest observed concentration in the linear range of the calibration curve. The linear range and the detection limit for the simultaneous determination of UA, XA and HX at poly l-methionine/GCE were compared with the recently reported chemically modified electrodes in Table 2. According to Table 2, present work has lower LOD than that of other works. 3.10. Interference study
Fig. 6. Cyclic voltammograms of the phosphate buffer solution (pH 7.20) containing 50 M UA (I), 20 M XA (II) and 60 M HX (III) at bare GCE (a) and poly l-methionine/GCE (b) at scan rate 100 mV s−1 .
For evaluating selectivity of the poly(l-methionine)/GCE modified electrode, various possible interfering species were examined for their effects on the determination of UA, XA and HX (UA = 50 M, XA = 20 M and HX = 60 M). The results listed in Table 3. The recovery percent of UA, XA and HX were in the range 98.21–104.54%, which indicates that those species have no interference effects on the determinations of analytes. As you can see in Table 3, values of RSD (%) for UA, XA and HX were under 3%, indicating that the method has good accuracy.
3.9. Linear range, detection limits and limit of quantification 3.11. Reproducibility and stability of the modified electrode The Linear range, detection limits (LOD) and limit of quantification (LOQ) were individually studied using differential pulse voltammetry (DPV) in pH 7.20 phosphate buffer. Since DPV has a higher current sensitivity and better resolution than CV, it was used for simultaneous determination of UA, XA and HX. Fig. 9
In order to test the repeatability of the poly(l-methionine)/GCE modified electrode, the DPV for 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution were determined repeatedly at the identical surface of poly(l-methionine)/GCE. The intra-day
Fig. 7. Cyclic voltammograms of UA in 0.1 M phosphate buffer (pH 7.20), at various scan rates: a–g show 20, 40, 60, 80, 100, 150 and 200 mV s−1 , respectively. Insets: (A) variations of Ip versus scan rates and (B) Tafel plot at a scan rate of 20 mV s−1 .
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Fig. 8. Chronoamperograms obtained at PLMT/GCE in 0.1 M phosphate buffer solution (pH 7.20) for different concentrations of XA. The numbers 1 → 5 correspond to 0.0, 0.1, 0.5, 0.75 and 0.95 M. Insets: (A) plots of I versus t−1/2 obtained from chronoamperograms 2 → 5 and (B) plot of the slope of the straight lines against the XA concentration.
Fig. 9. Differential pulse voltammograms of a fixed concentration of UA (1.0 mM) and XA (1.0 mM) and different concentrations of HX: (a) 0.02, (b) 0.04, (c) 0.06, (d) 0.1, (e) 0.2, (f) 0.4, (g) 0.6, (h) 0.8 and (i) 1 M in 0.1 M phosphate buffer solution (pH 7.20) at the PLMT/GCE. Inset: analytical plot for HX voltammetric determination obtained from its DPV data. (In all the calibration plots, each data point is the average of three current values.)
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Table 3 Effect of different interferents on the voltammetric signal of UA, XA and HX at the PLMT/GCE. Interfering agent
Dopamine Ascorbic acid Epinephrine
Interfering agent
80 1000 100
Recovery (%)
RSD (%)
UA
XA
HX
UA
XA
HX
100.00 104.54 100.00
102.22 98.21 99.23
100.00 103.33 98.87
1.5 2.1 1.7
1.1 1.6 0.8
2.7 1.3 1.5
Fig. 11. Differential pulse voltammograms of UA, XA and HX in human serum samples (a) blank serum sample was attenuated 10-fold with pH 7.20 phosphate buffer solutions; (b) serum sample spiked with 0.02 M UA, XA and HX at scan rate 30 mV s−1 .
Fig. 10. Differential pulse voltammograms of UA (50 M), XA (30 M) and HX (40 M) in 0.1 M phosphate buffer solution with pH 7.20 at the PLMT/GCE, (a) one day, (b) after a week and (c) after ten days at a scan rate of 30 mV s−1 .
the laboratory, the modified electrode retains 97.56%, 98.08% and 99.60% for UA, XA and HX, respectively of its initial response after a week and 91.46%, 90.38% and 92.40% after 10 days. The above results demonstrated that the proposed method had good repeatability, excellent precision and long-term stability. 3.12. Analytical applications
precision of the method was evaluated by repeating five experiments in the same solution containing 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution taken separately using the same poly(l-methionine)/GCE modified electrode. The RSD was found to be 1.25%, 1.92% and 1.6% for UA, XA and HX, respectively, indicating excellent reproducibility of the modified electrode. Further, inter-day precision was investigated by measuring the current response of the modified electrode for 7 consecutive days for the same concentration of 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution taken separately and the respective relative standard deviations were found to be 1.81%, 1.12% and 1.74%. Thus, it demonstrated the good reproducibility of the method using the poly(l-methionine)/GCE modified electrode. The same solutions were also determined with 5 modified electrodes made independently, and the RSD for peak currents of UA, XA and HX were 1.85%, 1.47% and 1.85%, respectively. In order to test the stability of the poly(l-methionine)/GCE modified electrode, the DPVs for 50 M UA, 30 M XA and 60 M HX in 0.1 M PBS (pH 7.20) solution were determined repeatedly at the identical surface of poly(l-methionine)/GCE (Fig. 10). When the electrode is stored in
The analytical utility of the modified electrode was illustrated by simultaneous determination of UA, XA and HX in human serum samples. The serum samples were diluted 10-fold with pH 7.20 phosphate buffer solution and the three compounds were determined simultaneously by the DPV method. Curves a and b in Fig. 11 represent differential pulse voltammograms for blank serum sample and a spiked serum sample containing standard solution of UA (2.0 × 10−8 M), XA (2.0 × 10−8 M) and HX (2.0 × 10−8 M), respectively. The DPV of serum sample (Fig. 11a) showed three oxidation peaks at 0.23 V, 0.59 V and 0.95 V, respectively. To confirm the observed oxidation peaks are due to the oxidation of UA, XA and HX, the serum sample was spiked with known concentrations of UA, XA and HX. The current response at 0.24 V, 0.59 V and 0.95 V enhanced after the addition of UA, XA and HX (Fig. 11b), which demonstrated that the observed oxidation peaks are due to the oxidation of the three compounds. For the recovery, all the serum samples were diluted 100-fold with pH 7.20 phosphate buffer solutions. The concentrations of UA, XA and HX were determined using the standard addition method, and the results are listed in Table 4.
Table 4 Simultaneous determination of UA, XA and HX in serum sample. No. Exp.
1 2 3
UA spiking, 10−7 M
XA spiking, 10−7 M
HX spiking, 10−7 M
UA found, 10−7 M
XA found, 10−7 M
HX found, 10−7 M
0.5 0.6 0.5
0.3 0.4 0.3
0.2 0.5 0.2
0.51 0.58 0.52
0.32 0.41 0.30
0.22 0.50 0.20
Recovery (%)
RSD (%)
UA
XA
HX
UA
XA
HX
101.4 97.3 104.0
106.7 102.7 100.9
109.0 99.4 99.0
1.7 0.9 1.9
1.8 1.5 2.1
1.2 1.6 2.4
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It can be seen that all spike recoveries were accurate and precise, which indicated that the proposed method could be efficiently used for simultaneous detection of UA, XA and HX in real samples. 4. Conclusions In this work, the poly(l-methionine) modified electrode prepared by simple and fast electropolymerization method and we applied this electrode for the simultaneous determination of UA, XA and HX by cyclic voltammetry, chronoamperometry and differential pulse voltammetry. The results showed that the oxidation of UA, XA and HX is catalyzed at pH 7.20, where the peak potential of these compounds is shifted to less positive peak potentials at the surface of the PLMT. This electrode exhibits high selectivity, excellent sensitivity, good reproducibility, stability and accuracy in the voltammetric measurement of UA, XA and HX. Also, the modified electrode can be applied to the determination of purine derivatives in human serum samples with satisfactory results. References [1] H. John, T. Ayvazian, S. Skupp, The study of purine utilization and excretion in a xanthinuric man, Journal of Clinical Investigation 44 (1965) 1248–1260. [2] T. Yamamoto, Y. Moriwaki, S. Takahashi, Effect of ethanol on metabolism of purine bases (hypoxanthine, xanthine, and uric acid), Clinica Chimica Acta 356 (2005) 35–57. [3] N.V. Bhagavan, Medical Biochemistry, fourth ed., Academic Press, USA, 2002. [4] J.S.N. Dutt, M.F. Cardosi, J. Davis, Electrochemical tagging of urate: developing new redox probes, Analyst 128 (2003) 811–813. [5] 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, Journal of Electroanalytical Chemistry 642 (2010) 135–142. [6] 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. [7] E.E. Kelley, A. Trostchansky, H. Rubbo, B.A. Freeman, R. Radi, M.M. Tarpey, Binding of xanthine oxidase to glycosaminoglycans limits inhibition by oxypurinol, Journal of Biological Chemistry 279 (2004) 37231–37234. [8] M. Czauderna, J. Kowalczyk, Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine urine by high-performance liquid chromatography and photodiode array detection, Journal of Chromatography B: Biomedical Sciences and Applications 744 (2000) 129–138. [9] 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, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences 837 (2006) 1–10. [10] F. Carlucci, A. Tabucchi, B. Biagioli, G. Sani, G. Lisi, M. Maccherini, F. Rosi, E. Marinello, Capillary electrophoresis in the evaluation of ischemic injury: simultaneous determination of purine compounds and glutathione, Electrophoresis 21 (2000) 1552–1557. [11] E. Caussé, 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, Electrophoresis 28 (2007) 381–387. [12] A.M. Cubukcu, S. Timur, U. Anik, Examination of the performance of the glassy carbon paste electrode modified with gold nanoparticle and xanthine oxidase for xanthine and hypoxanthine detection, Talanta 74 (2007) 434–439. [13] G. Park, R.N. Adams, W.R. White, A rapid accurate electrochemical method for serum uric acid, Analytical Letters 5 (1972) 887–896. [14] R.N. Goyal, A. Mittal, S. Sharma, Simultaneous voltammetric determination of hypoxanthine, xanthine and uric acid, Electroanalysis 6 (1994) 609–611. [15] Y. Wang, Simultaneous determination of uric acid, xanthine and hypoxanthine at poly(pyrocatechol violet)/functionalized multi-walled carbon nanotubes composite film modified electrode, Colloids and Surfaces B: Biointerfaces 88 (2011) 614–621. [16] D. Sun, Y. Zhang, F. Wang, K. Wu, J. Chen, Y. Zhou, Electrochemical sensor for simultaneous detection of ascorbic acid, uric acid and xanthine based on the surface enhancement effect of mesoporous silica, Sensors and Actuators B: Chemical 14 (2009) 641–645. [17] J.M. Zen, Y.Y. Lai, H.H. Yang, A.S. Kumar, Multianalyte sensor for the simultaneous determination of hypoxanthine, xanthine and uric acid based on a preanodized nontronite-coated screen-printed electrode, Sensors and Actuators B: Chemical 84 (2002) 237–244. [18] C.W. Xu, R. Zeng, P.K. Shen, Z.D. Wei, Synergistic effect of CeO2 modified Pt/C catalysts on the alcohols oxidation, Electrochimica Acta 51 (2005) 1031–1035. [19] T. Inoue, J.R. Kirchhoff, Electrochemical detection of thiols with a coenzyme pyrrologuinoline quinone modified electrode, Analytical Chemistry 72 (2000) 5755–5760.
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Biographies Reza Ojani is a full professor of analytical chemistry, Mazandaran University, Babolsar, Iran. He received BSc from Gilan University, 1988; MSc from Tabriz University in analytical chemistry, 1991; and PhD from Tabriz University in analytical chemistry, 1996. His research interests cover electrochemical sensors, biosensors, electrochemical behavior of nanoparticles and fuel cell.
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Ali Alinezhad received the BSc degree in applied chemistry from Tabriz University, Iran, in 1996; MSc from Mazandaran University in analytical chemistry, 1999; and PhD from Mazandaran University in analytical chemistry, 2008. His research interests electrocatalyze, modified electrodes, electrosynthesis and electrochemistry in aqueous environments micelle.
Zahra Abedi received the BSc degree in applied chemistry from Payam Noor University, Behshar, Iran, in 2008. Now, she is working toward the MSc degree at Mazandaran University, Babolsar, Iran. Her interests are analytical chemistry, bioelectroanalytical chemistry and electrochemical properties of nanomaterials.