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PAPER

Graphitized mesoporous carbon modied glassy carbon electrode for selective sensing of xanthine, hypoxanthine and uric acid
Rajendiran Thangaraj and Annamalai Senthil Kumar*
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Received 10th January 2012, Accepted 19th April 2012 DOI: 10.1039/c2ay25029b An efcient electrochemical sensor for simultaneous electrochemical sensing of three purine compounds, uric acid (UA), xanthine (X) and hypoxanthine (HX), using a graphitized mesoporous carbon (GMC) modied glassy carbon electrode (GCE/GMC) has been demonstrated in pH 7 phosphate buffer solution without any enzyme, prior to electrode activation, surfactant and sample pre-concentration step. Electrochemical investigation of the GCE/GMC with [Fe(CN)6]3 indicates metallic conductor like surface features of the modied electrode. A diffusion controlled reaction mechanism was identied for the electro-oxidation of the three purine compounds with an electrocatalytic pathway, except for the UA, where it shows a surface area effect with a mixed-diffusion and adsorption controlled mechanism at higher scan rates (>70 mV s1). Calculated full-width of the half maximum values for the simultaneous detection of the three purine compounds are 42, 53 and 64 mV respectively and these are the lowest values ever reported in the literature, suggesting effective electron-transfer behaviour of the modied electrode for the purine oxidations. Calibration plots for the simultaneous detection of the purine compounds were linear in the concentration range of 20 400 mM, 20320 mM and 20240 mM for UA, X and HX with detection limit values of 110 nM, 388 nM, and 351 nM respectively. Selective sensing of the purine compounds in human blood-plasma, urine and sh samples was successfully demonstrated with recovery values $100%.

1. Introduction
Xanthine (X), hypoxanthine (HX) and uric acid (UA) are important purine bases, where X and HX are the intermediates and UA is an end product of the purine metabolism of the cells of humans, animals and plants.13 When cells die, the purines in their genetic material break down. Purine catabolism of a dead sh is as follows: adenosine triphosphate / adenosine diphosphate / adenosine monophosphate / inosine monophosphate / inosine / HX / X / UA.13 Xanthine oxidase (XOD) is the specic enzyme present in the biological system, which helps to catalyze the HX and UA oxidation reactions.4 Analyses of the intermediates and the end product can give vital information about the freshness of the sh foods.4 For example, muscles of the freshly dead sh contain higher concentrations of HX and X than the end product, UA; while for the aged and decomposed sh, the concentration of UA is higher than that of X and HX.5 Similarly, abnormalities of the

Environmental and Analytical Chemistry Division, School of Advanced Sciences, Vellore Institute of Technology University, Vellore-632 014, India. E-mail: askumarchem@yahoo.com; Fax: +91-416-2243091/93; Tel: +91-416-2202754 Electronic supplementary information (ESI) available: Effect of solution pH on simultaneous analysis of three purine bases at GCE/GMC (Fig. S1). See DOI: 10.1039/c2ay25029b

purine compounds present in humans are the clinical symptoms for some critical diseases. For instance, increased excretion of the hypoxanthine and xanthine in urine is the indication of the inherited disease, Xanthinuria.6,7 Uric acid also serves as an antioxidant and helps to prevent damage to our blood vessel linings.8 For healthy individuals, the concentration of the UA present in the plasma is approximately 116500 mM and in the range of 1.24.5 mM in urinary excretion.9 In diseases such as gout, hyperuricemia and LeschNyhan syndrome, high levels of the UA are found in the blood samples.10 Meanwhile, relatively few foods contain concentrated amounts of the purines with high-protein content. Examples are: organ meats like kidney, sh, herring, sardines, mussels and yeast.11 Hence selective, sensitive and simultaneous sensing of UA, X and HX in biological, food and clinical samples is of signicant research interest in analytical chemistry. Conventional analytical methods available for the purine analysis are based on separation (HPLC or capillary electrophoresis) coupled with UV-Vis or uorescence spectroscopic techniques.1220 These methods are time consuming and require several off-line pre-concentration steps for the real sample analyses. Several electrochemical methods are available for the separation-less and enzyme-less analysis of the three purine compounds. Following are the recently reported working electrodes for the simultaneous detection of the three compounds:
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unmodied glassy carbon electrode (GCE),21 preanodized carbon paste electrode22 and functionalized single walled carbon nanotubecarbon paste electrode23 in acid pH solutions, and Naon modied lead-ruthenate pyrochlore,24 Ru(DMSO)4Cl2 electrodes,25 preanodized nontronite clay coated screen-printed electrode,26 electropolymerized poly(bromocresol purple) modied GCE,27 and impure multi-walled carbon nanotube (MWCNT).28 Most of the reported procedures either require expensive chemicals or require complicated electrode preparation steps which include extensive pre-anodization of the underlying or modied electrode and the use of expensive starting materials such as Naon, carbon nanotubes and ruthenium.24 Few chemically modied electrodes were also reported for the simultaneous detection of two purine compounds (UA and X) with examples being: 2-amino-1,3,4,thiadiazole modied GCE,29 mesoporous SiO2graphite paste electrode30 and puried MWCNTdihexadecyl hydrogen phosphate surfactant modied electrode.31 Recently, a XOD enzyme biosensor constituted with functionalized graphene oxide + conducting polypyrrole graft copolymer + poly(styrenesulfonic acid-g-pyrrole) modied electrode was reported for HX sensing in a physiological solution.32 In this work, we are reporting a commercially received graphitized mesoporous carbon (GMC) modied GCE (designated as GCE/GME) prepared by a much simpler method than the previously reported procedures without any enzyme, surfactant and activation or functionalization of the electrode. The GCE/GMC is highly useful for a sensitive, selective and simultaneous voltammetric detection of the three purine compounds UA, X and HX in a physiological solution. Mesoporous materials in general have pores with an average diameter size (250 nm) in between the range of micro-porous (<2 nm) and macro-porous (>50 nm) materials. GMC is a porous carbon containing surface graphitic frameworks, prepared using certain soft polymers as a precursor and mesoporous silica materials (MSM) as a template by a high temperature pyrolysis procedure (non-conventional method of preparation).33 Structural features of the GMCs include homogeneity with signicant graphite-like domains and stacking heights, low amounts of surface functional groups, and low occurrences of imperfections such as twists, non-aromatic links, and carbon valencies.34 GMC has been demonstrated as a highly efcient support for Pt catalyst immobilization and for the enhanced direct methanol oxidation and oxygen reduction reactions suitable for fuel cell applications.35,36 Some of the conventional carbons, which are rich in carbonyl, phenolic and alcoholic functional groups, often result in severe oxidative corrosion during the electro-catalysis.37,38 But no such complications were reported with the GMC. The GMC has been used as a catalyst for several organic functional group transformations including dehydrogenation of isobutane.39 The GMC material was rarely used for chemically modied electrode preparation and electroanalytical applications.40 Interestingly in this work, the GCE/GMC showed an efcient sensing of the three purine compounds without any ascorbic acid (AA) interference in a physiological solution. Finally, the modied electrode was successfully applied for simultaneous or selective sensing of the purine compounds in different kinds of clinical (blood and urine) and sh samples.
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2. Experimental
2.1. Chemicals GMC (50 nm and 99.95% purity), MWCNT (outer diameter: 10 15 nm; inner diameter: 26 nm, length 0.110 mm and $90% purity), uric acid, xanthine and hypoxanthine and ascorbic acid were of analytical grade and purchased from Sigma-Aldrich. They were used as received without any further purication. Aqueous solutions were prepared using deionized and alkaline KMnO4 distilled water (designated as DD water). Phosphate buffer solution (PBS, pH 7.1 0.05) of 0.1 M ionic strength was used as a supporting electrolyte. 2.2. Measurements

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Voltammetric measurements were carried out using a CHI660C Electrochemical work station (USA) with 10 mL working volume. The electrode system consists of a glassy carbon electrode (GCE) and its chemically modied electrode (CME) as a working electrode (0.0707 cm2), Ag/AgCl (3 M KCl) as a reference electrode and platinum wire as a counter electrode. The surface of the GCE was cleaned both mechanically (polished with 0.05 micron alumina powder in a BAS polishing kit, cleaned with acetone and washed with double distilled water) and electrochemically by performing cyclic voltammetry (CV) continuously for 20 cycles in a potential window of 0.2 to 1.2 V vs. Ag/AgCl in PBS. The GMC surface morphology was examined by transmission electron microscopy (TEM, JEOL-3010). A Bruker D8 Advanced diffractometer X-ray diffraction (XRD,  was Germany) instrument with Cu Ka source (l 1.5418 A) used to probe the crystallographic information of the GMC. 2.3. Preparation of the GCE/GMC modied electrode

A GMCethanol suspension stock was rst prepared by mixing 1 mg of the GMC powder with 500 mL of absolute ethanol (99.9%) followed by 10 minutes sonication at room temperature (T 300(2) K). For the GCE/GMC preparation, 5 mL of the stock was drop coated on the cleaned GCE surface and air dried for 10 minutes at room temperature. The procedure allows for preparing a near uniform layer of GMC on the GCE surface. The as-prepared GMC layer was further electrochemically pretreated by continuous potential cycling (n 20, n no. of cycles) in the potential window of 0.2 to 0.8 V vs. Ag/AgCl at a scan rate (v) of 50 mV s1 in pH 7 PBS. The GCE/GMC did not show any redox feature in the blank base electrolyte solution. Similar to the above procedure, GCE/MWCNT was also prepared by a drop casting methodology. Since dissolved oxygen did not inuence the electrochemical response, experiments were all carried out without any deaeration, as a physiological solution. The differential pulse voltammetric (DPV) technique was used as a quantitative electrochemical tool for the simultaneous detection of the purine bases X, HX and UA in pH 7 PBS. 2.4. Real samples preparation

Human blood plasma (#1(A)a, #1(A)b, #1B), healthy human urine (#2#4), red blood cell (RBC) and pus cell contaminated urine (#5#7) and fresh sh esh (#8#11) samples were
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subjected to real sample analyses. Three healthy blood samples of volume 10 mL, where two samples belonging to the same person were collected at 60 day time intervals (#1(A)a and b) with the help of a staff nurse from the Vellore Institute of Technology (VIT) health care centre. The three healthy human urine samples (#2 #4) and three RBC/pus cell contaminated urine samples (#5#7) were also collected from the VIT university health care centre. The RBC/pus cell counts in the urine samples are 1015/510, 1015/ 1015 and 1520/36 respectively for samples #5, #6 and #7. Prior to the analysis, the urine samples were ltered with ordinary lter paper and diluted (dilution factor 100) using PBS. Fresh sh samples: seer sh #8 (Scomberomorus guttatus), red snapper #9 (Himantura bleekeri), goldstripe sardinella #10 (Sardinella gibbosa), and carp sh #11 (semisulcatus) were purchased from a local animal food market in the Vellore city. All these samples were stored in a refrigerator at $5  C until use. Following is the procedure for the blood plasma sample preparation: 10 mL of the blood sample with a pinch of ethylenediamine tetraacetic acid (EDTA) was rst centrifuged at 1600 rpm for about 20 minutes and the concentric plasma that appeared as a supernatant was taken for further analysis after proper dilution with PBS (dilution factor 5). For the sh real sample preparation, about 2 g of the sh esh was weighed and homogenized with 10 mL of PBS using a mortar and pestle. The homogenized solution was ltered and further diluted with PBS (dilution factor 5). For all the real sample analyses, a 10 mL aliquot was taken and examined with the differential pulse voltammetric (DPV) technique. A standard addition approach was adopted for the quantication of the purine concentrations in the real samples. Since there is a weak adsorption of xanthine on the GCE/GMC surface, after the experiment with the analyte, the electrode was removed from the cell and immersed in 10 mL blank PBS for 10 seconds at room temperature. Such an experimental procedure gives easy regeneration of the GCE/GMC for the successive electroanalytical measurements.

The XRD pattern of the GMC was compared with another well-known crystalline carbon, MWCNT. Fig. 2 shows a well dened and intense diffraction peak at 2q 26.05 along with a weak peak at 43.02 that were obtained from the XRD of the GMC powder sample. The values correspond to the hkl index of {002} and {101} respectively for the hexagonal graphitic structure on the mesoporous carbon. The control XRD pattern of the MWCNT material also shows similar 2q values at 26.07 and 43.13 . The calculated d-spacing (d002) value for the {002} peak using the Braggs equation, d002 nl/2sin(q), where d002 interplanar distance, l wavelength and q angle of diffraction,  for both the GMC and MWCNT samples. The values is 3.41 A quoted in the literature for the unmodied mesoporous and graphitized mesoporous carbon (GMC) materials are 3.71 and  respectively.35,41 The 0.34 A  reduction in the d-spacing is 3.36 A an indication of the enhanced crystallinity of the GMC compared to the MWCNT. 3.2. Electrochemical characterization of the GCE/GMC

The ferricyanide/ferrocyanide redox couple is a bench mark one electron-transfer electrochemical system, useful to access the conductive and the electron-transfer features of a modied electrode system. Fig. 3(A)a is the CV response of the GCE/ GMC electrode in the presence of 1 mM of potassium ferricyanide at a scan rate of 10 mV s1. A well dened redox couple with anodic (Epa) and cathodic (Epc) peak potential values at 240 and 155 mV vs. Ag/AgCl respectively was noticed.

3. Results and discussion

3.1. Physicochemical characterization of the GMC TEM images of the GMC at different magnications are given in Fig. 1AC. Randomly spaced empty clusters of uniform size $53 nm (outer diameter) are seen in the photographs. The black spots observed in the photographs are agglomerated units of the GMC. Details of the graphitic layers could be identied from the XRD analysis.

Fig. 2 XRD patterns of graphitized mesoporous carbon and a multiwalled carbon nanotube.

Fig. 1 TEM images of GMC at different magnications.

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3.3. Electrochemical and catalytic behaviours of the GCE/ GMC Fig. 4AC show comparative CV responses of the GCE/GMC and unmodied GCE with UA, X and HX discretely at a scan rate of 50 mV s1 in pH 7 PBS. As can be seen from the CV of UA, the unmodied GCE shows a marked anodic oxidation peak at 295 mV vs. Ag/AgCl. The GCE/GMC also showed a similar peak potential value but with two and half times enhancement in the anodic peak current value over the GCE. The observation may be due to the surface area effect of the GCE/GMC. For the case of X (Fig. 4B), the GCE/GMC showed a profound oxidation peak at 650 mV and was three times higher in the peak current value than the unmodied GCE. Note that the X oxidation peak potential at GCE is 750 mV, which is 100 mV higher in over-potential than the GCE/GMC system. For the HX case (Fig. 4C), the unmodied electrode shows the absence of any CV oxidation peak up to 1100 mV, whereas the GCE/GMC yielded a clear oxidation peak at 980 mV. In general, the surface area effect can be referred as when the modied electrode showed an enhanced oxidation/reduction current response to the analyte over its corresponding unmodied electrode without altering the oxidation/reduction peak potential.42 On the other hand if the peak potential is altered, for example, the analyte oxidation potential is signicantly reduced by the modied electrode (over-potential reduction with the symbol h) and it is then referred to as the electrocatalytic effect. In our case, X and HX have shown signicant reductions in the h values, while for the case of the UA there is no such reduction in the h; however, only enhancement in the peak current value was noticed. Based on the information it can be concluded that X and HX oxidations are due to the electrocatalytic behaviour, while for the UA oxidation referred, it is due to the surface area effect of GCE/GMC. In continuation of the CV studies, DPV was also performed on the above purine compounds comparatively with GCE and GCE/GMC as in Fig. 4DF. All the three purine compounds show well-dened and sharp peak currents respectively at 250, 580 and 900 mV vs. Ag/AgCl for UA, X and HX on the GCE/ GMC. Unlike the CV behaviours, DPV of the unmodied GCE shows signicant decreases in the peak current signals and high oxidation potential values, 460 and 780 mV for the UA and X respectively, and nil response to HX. Calculated h reductions for the UA and X are 200 and 220 mV respectively, in the DPV. But, no such observations were noticed in the CV (Fig. 4AC). Presumably, the applied amplitude (50 mV) and pulse effects (pulse width 0.2 s; pulse period 0.5 s) may have some hindrance to the electro-oxidation of the purine compounds on the GCE, while for the case with GCE/GMC a positive effect by the DPV parameters and hence considerable sharp peaks and reduction in the h were observed. Hence, it is a clear advantage of using DPV as the qualitative tool for sensitive analyses of the compounds in this work. Note that we have optimized the DPV parameters viz., pulse width, amplitude and pulse period with respect to the GMC modied electrode in order to get sharp voltammetric responses to the analytes. This is the reason why the corresponding unmodied GCE showed broad peaks in Fig. 4D and E.
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Fig. 3 (A) CV responses of bare GCE and GCE/GMC with 1 mM of K3[Fe(CN)6] in 0.5 M KCl solution at v 10 mV s1. (B) Plot of ipa vs. v1/2 for the ferricyanide redox process at two different electrodes.

Calculated peak-to-peak potential (DEp Epa Epc) and apparent standard electrode potential (Eo0 Epa + Epc/2) values are 85 mV and 198 mV vs. Ag/AgCl respectively. For comparison, the unmodied GCE is also subjected to the ferricyanide experiment as in Fig. 3(A)b. Corresponding DEp and Eo0 obtained on the GCE are 86 and 200 mV vs. Ag/AgCl. The similarity in the DEp and Eo0 values between the GCE and GCE/GMC indicates appreciable electron-transfer behavior of the GCE/GMC closer to the metallic conductive electrode. From the reversible redox reaction, the electrochemically accessible surface area, Ae (cm2), value could be calculated using the standard RandleSevcik equation: ipa or ipc 2.69 105AeD1/2n3/2v1/2C, where n is the number of electrons involved in the redox reaction, D is the diffusion coefcient (7.6 106 cm2 s1) and C is the concentration of the redox couple. Fig. 3B shows the plots of ipa vs. v1/2 for the ferricyanide at GCE and GCE/GMC. The GCE shows a linear line up to v 500 mV s1, while for the GCE/GMC the linearity is maintained up to 70 mV s1, thereafter the line tends to saturate. The calculated slope (ipa/v1/2) value (1.32 mA mV1/2 s1/2) is the same for the GCE and GCE/GMC. After substitution of the slope value in the RandlesSevcik equation, the Ae value is calculated to be 0.0562 cm2, which is equal to $80% of the geometric surface area of the working electrode (0.0707 cm2). Note that GCE is atomically smooth and possesses near ideal surface features, whereas the GCE/GMC is the modied electrode with graphitic and non-graphitic porous carbon units. Even though the GMC has high surface area (BET surface area 900 m2 g1),33,34 in consideration with the Ae values, it can be speculated that only part of the total surface area of the electrodes is accessible for the electron-transfer reaction. The nature of surface polishing methods including the mechanical polishing of the electrode and electrochemical pretreatments can change the surface area of the GCE. On the other hand, it is expected that there will be some diffusion restriction of the reduced and oxidized forms of the ferrocyanide complex within the GMC. This observation could be reected by an improper (non-ideal) tail-off peak current and non-linearity in the ipa vs. v1/2 plot at v > 70 mV s1 for the redox couple on the GCE/GMC working electrode, unlike the GCE case (Fig. 3(A)b). Nevertheless, due to the unique structural feature of the GMC, the electrochemical response of the GCE/GMC is remarkable and effective over the unmodied electrode for the purine compounds oxidation.
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Fig. 4 Discrete CV (AC) and DPV (DF) responses of the electrochemical oxidations of UA, X and HX at GCE and GCE/GMC in a pH 7 PBS. DPV parameters are: amplitude 50 mV; increment potential 4 mV; pulse width 0.2 s; pulse period 500 s.

In order to compare the efciency of the electron-transfer behaviours (and in turn to the sharp peak currents), DPV peaks full-width of the half maximum (FWHM) value is taken as a sensitive parameter. Calculated FWHM values are 42, 53 and 64 mV respectively for the UA, X and HX. The FWHM values for the recently reported Ru(DMSO)4Cl2Naon modied electrode are 75, 75 and 100 mV respectively.25 The lowered FWHM values with the GCE/GMC are the clear evidence for the efcient peak current feature of the present system. Simultaneous analysis of the UA, X and HX on a GCE/GMC was next examined by CV and DPV techniques as in Fig. 5A and B respectively. Interestingly, the GCE/GMC shows well-dened and well-separated oxidation peak current responses. But the unmodied GCE displays a 13-fold decrease in the voltammetric peak current at the oxidation potentials of 400 and 800 mV for the UA and X, and an absence of HX signal. The current signals are ill dened too. Appearance of these current signals may be due to the response of overlapped UA, X and HX analytes. Calculated FWHM values for the analyte peaks from the simultaneous DPV

Fig. 5 Simultaneous CV (A) and DPV (B) responses of GCE and GCE/ GMC for a mixture of UA, X and HX dissolved in pH 7 PBS. CV scan rate (v) 50 mV s1. Other DPV parameters are as in Fig. 4.

measurements are UA 54, X 50 and HX 50 mV and these values were considerably smaller than the previously reported values of 59, 74, 86 mV on Ru(DMSO)4Cl2Naon,25 and 65, 70, 140 mV on poly(bromocresol purple) modied electrodes for the simultaneous oxidations of the three purines in a pH 7 PBS.27 All these experimental observations evidence the superior function of the GCE/GMC for the simultaneous oxidations of UA, X and HX in a physiological solution in this work. In order to check the nature of the electron-transfer mechanism, effects of CV scan rate (v) on the discrete oxidations of the three purine compounds were examined as in Fig. 6AC. An increase in the v implies a systematic increase in the peak currents for all the three compounds. Peak current values were measured and plotted against the scan rate (v) as a double logarithmic plot as in Fig. 6D. Calculated slope (vlog ipa/vlog v) values are: UA 0.3 (v < 70 mV s1) and 0.85 (v > 80 mV s1), X 0.30 and HX 0.30. These values are closer to the ideal values of 0.5 and 1 corresponding to diffusion and adsorption controlled electron-transfer mechanisms.43 Since the X has some weak adsorption effect on the GCE/ GMC (see the Experimental section), a slope value near 1 would be expected rather than $0.5 in this work. But the measured experimental value is $0.5, which indicates a diffusion controlled mechanism of the adsorbed X within the GCE/GMC lm. The trend in the mechanism of the UA changes from diffusion to adsorption controlled reaction at scan rate >70 mV s1 (slope 0.85), which resembles the trend of the ferricyanide case (Fig. 3A). Presumably, like ferricyanide, there will be some diffusion restriction for the UA within the pores of the GMC at a high scan rate, which in turn leads to change in the reaction mechanism. Exact details for the occurrence of such reaction are still unknown. On the basis of Epa ([balog(v)]/2) + constant, for a totally irreversible diffusion-controlled process44,45 the ba (i.e., Tafel slope) value is measured as 10, 40 and 43 mV per decade for UA, X and HX oxidation reactions respectively on a GCE/GMC (Fig. 6E). Assuming one-electron transfer in the rate
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Fig. 6 Effect of CV scan rate from 5500 mV s1 on the electrochemical oxidation of (A) UA, (B) X and (C) HX at GCE/GMC in a pH 7 PBS. Plots of log(ipa) vs. log v (D) and Epa vs. log v (E) for the purine electrochemical oxidation reactions. (F) Typical illustration for the electrochemical oxidations of the three purines on the GCE/GMC.

determination step (i.e., na 1), since ba 2.303RT/naFaa, the anodic-transfer coefcient (aa) can then be calculated as 5.9, 1.48 and 1.37 for the UA, X and HX respectively. Previously, Zen et al. reported an aa value of $1 for an irreversible oxidation of cysteine on a lead-ruthenate pyrochlore modied electrode.46 Hence, the high values obtained in the present work are an indication of the effective catalytic performance of the GCE/ GMC to the purine oxidation reactions. Fig. 6F is a typical illustration for the electrochemical oxidation of the three purine compounds on the GCE/GMC. Fig. S1(A) shows the effect of solution pH (in the range pH 3 8) on the simultaneous oxidations of the three analytes by the DPV. Plots of the respective anodic peak current and peak potential against the solution pH are displayed in Fig. S1(B) and S1(C) respectively. The peak current values increased positively against the solution pH and attained the maximum at pH $7. The peak potential values were systematically decreased against increase in the pH values. A slope value of 60 mV per pH was calculated for all the three compounds on the GCE/GMC, suggesting the proton-coupled electron-transfer mechanism with equal number of proton/electron involvement in the reaction pathway (Nernstian case). Usually, depending on the pKa value of an analyte, there is a marked alteration in the trend of the oxidation peak potential value.24 Unfortunately, considering the pKa values of UA (5.4 and 10.3), X (7.4 and 11.2) and HX (8.94 and 12.10), there were no alterations in the peak potentials with respect to the pKa values in this work. This observation further suggested that the protonation reaction is not a rate determining step in the electrochemical oxidation processes. 3.4. Analytical measurements

Effects of UA, X and HX concentrations (calibration) on the simultaneous detection of the individual analytes are next studied
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by systematically increasing one analyte concentration with the other two xed as in Fig. 7AC. The optimal DPV parameters used in this work are: increment 4 mV, amplitude 50 mV, pulse width 0.2 s, pulse period 0.5 s and sampling width 0.0167 s. Interestingly, systematic variations in the peak currents upon increases in the analyte concentrations were noticed. As seen in Fig. 7A, DPV of the UA shows regular increases in the peak current signals up to 400 mM with current sensitivity and regression coefcient values of 75.3 nA mM1 and 0.996 respectively. Ten repetitive measurements of 25 mM of UA result in a relative standard deviation (RSD) value of 0.88%. For the UA, the calculated detection limit (S/N 3) value is 110 nM. Analytical parameters for the X are: linearity up to 320 mM; sensitivity 61.8 nA mM1; regression 0.988, RSD 1.29% ([X] 25 mM, n 10) and detection limit 388 nM. Similarly for HX, the results are: linearity up to 240 mM; sensitivity 112.7 nA mM1; regression 0.996, RSD 1.87% ([HX] 25 mM, n 10) and detection limit 351 nM. Obtained detection limit values in this work are comparable to or even better than those previously reported in the literature. For instance, reported detection limit values for UA, X and HX on a nontronite clay coated screen-printed electrode with 2 V pre-anodization and 40 s of pre-concentration are 0.42 mM, 7.0 mM and 0.34 mM,26 poly(bromocresol purple) modied GCE; 0.20 mM, 0.06 mM and 0.12 mM,27 NaonRu(DMSO)4Cl2; 0.37 mM, 2.35 mM and 2.37 mM,25 and impure MWCNT; 0.141 mM, 0.134 mM, and 2.87 mM,28 respectively. Simple preparation method and using relatively low cost carbon materials (other than the MWCNT) as a working electrode without any enzyme, activation and preconcentration procedures are the superior features of the present working electrode over the existing CMEs. Note that the advantage of elimination of the ascorbic acid interference, unlike the MWCNT, was achieved with the GMC material (see Section 3.5). With regard to a stable response of the GCE/GMC surface,
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Fig. 7 Typical DPV responses for the simultaneous detection of UA, X and HX using a GCE/GMC in a pH 7 PBS. Plots of ipa vs. analyte concentration, [analyte], were given as the respective insets. DPV parameters are as in Fig. 4.

we expected that micro- and nano-porous voids might exist on the GCE surface (after the mechanical and electrochemical pretreatments); when GMC was modied as ethanol dispersion on the GCE surface, some of the portions of GMC might have occupied the porous sites of the underlying surface and thereby stabilizing the GCE/GMC. In order to check the reproducibility of the modied electrode, four modied electrodes were discretely prepared and subjected to DPV analysis of UA. The calculated RSD value for the peak currents is 3.06%, which indicates good reproducibility of preparation of the modied electrode.

Fig. 8 Effect of AA interference for the simultaneous DPV detection of the three xed purine compound concentrations on (A) GCE/MWCNT and (B) GCE/GMC in pH 7 PBS. Respective ipa vs. [AA] plots were given as insets in the gure.

3.5.

Interference study

With the aim to extend the GCE/GMC for blood plasma purines analyses, we have examined the effect of a co-existing biochemical, AA, on the simultaneous detection of the purine samples. For a comparison, GCE/MWCNT was also subjected to the AA interference study (Fig. 8A). In a whole blood sample, common interferences for the UA are glucose, cholesterol, triglycerides, proteins, sodium chloride and AA. All these compounds, except AA, are electro-inactive. As from the clinical diagnosis data, the concentration range of the UA and AA present in a healthy
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humans blood plasma is 112506 mM (2.08.5 mg dL1) and AA is 40 15 mM, respectively.4749 Fig. 8B is a typical DPV response of the GCE/GMC for the simultaneous measurements of a xed UA, X and HX content against variable AA concentrations in the range of 20500 mM. As can be seen, in the absence of AA, both GCE/MWCNT and GCE/GMC showed qualitatively similar DPV simultaneous responses. But in the presence of dilute AA, the GCE/MWCNT shows distinctly different voltammetric purine oxidation peaks. Plots of the three purine compounds anodic peak current (ipa, base-line corrected) against the AA concentration, [AA], are given as insets in Fig. 8A. Unfortunately, addition of a dilute concentration of AA 20 mM leads to about a three fold increase in the purine oxidation peak currents and these currents get saturated after that. With regard to the AA oxidation peak at 0.1 V vs. Ag/AgCl, a marked peak current signal was noticed only when [AA] > 100 mM. Below 100 mM AA, large background currents were observed instead of any AA oxidation feature. These observations ruled out the choice of the GCE/MWCNT for the precise simultaneous analyses of the purine compounds in
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Fig. 9 GCE/GMC responses for the real samples: (A) healthy human blood plasma samples (A)a and (A)b from the 1st and 60th day respectively, (B) healthy and (C) RBC/pus cell contaminated urine samples, and (D) a fresh sh sample in a pH 7 PBS. DPV parameters are as in Fig. 4.

the presence of dilute AA in a physiological solution. On the other hand, the GCE/GMC shows tolerable voltammetric current signals for the AA concentration up to 100 mM. In addition, the GCE/GMC did not show any AA oxidation peak current up to 100 mM (inset, Fig. 8B). Presumably the mesoporous structure has some negative interactive effects with the ve member non-aromatic cored AA. These observations evidence that GCE/GMC is the better choice for the detection of uric acid in human blood plasma. 3.6. Real sample analyses

Practical applications of the GCE/GMC for the simultaneous or individual analyses of the three purine compounds in human blood plasma, urine and sh esh samples were tested (Fig. 9 and

Table 1). Typical DPV responses of the GCE/GMC for healthy humans blood plasma samples collected at sixty day time intervals (1st day: #1(A)a and 60th day: #1(A)b), are displayed in Fig. 9(A)a and b. Real sample analyses data are presented in Table 1. A marked DPV peak at 0.25 V vs. Ag/AgCl was noticed corresponding to the presence of the UA in the blood plasma samples. Standard spikes of the UA result in a systematic increase in the peak currents on the real sample peak. This observation qualitatively conrms the presence of the UA in the blood plasma samples. Based on the standard addition method, the UA concentrations in the blood samples were calculated as 35.74 and 35.66 mM respectively for #1(A)a and #1(A)b. After correction of the dilution factor, the values turned out as 178.7 and 178.3 mM, which are in the range of the standard UA level.9 The observation of similar UA content values in those real

Table 1 Various purine real sample analyses using a GCE/GMC modied electrode by the standard addition method Detected value S. no. 1 2 3 4 5 6 7 8 9 10 11 12 13
a

Real samples Blood plasma (#1Aa) Blood plasma (#1Ab)a Blood plasma (#1B) Urine (#2) Urine (#3) Urine (#4) Urine (#5)c Urine (#6)c Urine (#7)c Fish (#8) Fish (#9) Fish (#10) Fish (#11)
a

Analyte UA UA UA X UA UA UA UA UA UA X X X X

Original 35.7 35.7 63.7 1.9 34.3 37.0 16.6 81.5 44.7 118.9 39.9 27.3 64.4 34.5

Totalb 178.5 mM 178.5 mM 318.5 mM 95.0 mM 3.4 mM 3.7 mM 1.7 mM 4.1 mM 2.2 mM 5.9 mM 199.5 mM (760 mg g1) 136.5 mM (520 mg g1) 322.0 mM (1200 mg g1) 172.5 mM (655 mg g1)

Added (mM) 20 20 20 20 30 30 20 50 50 50 30 60 30 25

Found (mM) 20.44 20.73 20.53 20.16 31.15 29.30 19.67 49.97 50.22 49.57 30.30 62.40 29.30 24.71

Recovery (%) 102.2 103.7 102.7 100.8 103.8 97.7 98.4 99.9 100.4 99.1 101.0 104.0 102.3 99.0

Collected from the same person. b After dilution factor correction. c Pus cell contaminated urine samples.

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samples proves the reliability of the present electrode for practical analyses. In another case, for healthy human urine, sample #2, two peaks at 0.34 and 0.68 V corresponding to the UA and X were observed (Fig. 9B). The standard addition method qualitatively conrms the presence of UA and X in the urine samples. Calculated UA and X concentration values are 318.7 and 96 mM respectively. In continuation, individual detection of UA present in two healthy humans urine samples (#3 and #4) was also examined and the values are 3.7 and 1.66 mM respectively and these values are within the standard UA limits only.9 Fig. 9B is a DPV of the RBC/pus cell containing urine sample #5. Entries 79 in Table 1 are the measured UA concentration values of the urine samples, #5#7. Since there is no precise literature available to refer the RBC/pus cell count versus UA level in urine, based on the experimental results we are concluding here that there is no dependence of the excreted UA level on the RBC/pus cell values of the human urine. Finally four fresh sh samples (#8#11) were subjected to the DPV analyses. The samples showed two peak responses at 0.6 and 0.95 V vs. Ag/AgCl corresponding to the X and HX compounds. All sh samples show ill dened HX peaks except sh sample #11. The standard addition of HX does not show increments in the peak current. We suspect some matrix effect with the HX analysis and hence it could not be detected for some kinds of sh samples. Nevertheless, the X analysis content in the sh is vital to judge the quality of the sh. As seen in Table 1, detected X contents were in the order of 1200 > 760 > 655 > 520 mg g1 for samples #10, #8, #11 and #9 respectively. The absence of UA with the animal food suggests appreciable freshness of the sh foods. Amongst the sh samples, sample #10 (Sardinella gibbosa) is found to be the richest content of X and hence these are the richest protein diet foods. Finally, calculated recovery values for the standard spikes of the purine samples all fall at $100%, which indicates the efciency of the GCE/GMC in the real sample analyses.

values for the analyses were $100%. Overall, the novelty of the present electrochemical sensor system lies in the simple preparation steps using relatively low cost GMC materials for the separation-less analyses of the three purine bases without any enzyme, the pre-concentration procedure, the pre-anodization steps and the extendibility of the sensor to various kinds of real samples.

Acknowledgements
The authors are grateful for the nancial support from the Department of Science and Technology (DST) under Technology System Development Programme, India. We also thank Material Science Division, Indian Institute of Technology, Madras for the TEM measurements.

References
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4. Conclusions
The GMC modied glassy carbon electrode was developed for efcient and simultaneous electrochemical sensing of three purine compounds: uric acid, xanthine and hypoxanthine in a physiological solution. The GMC modied electrode shows well dened well separated and very sharp purine oxidation signals at around 0.3, 0.6 and 0.95 V vs. Ag/AgCl respectively for the compounds. The purine oxidation mechanisms follow the electrocatalytic pathway for the X and HX, while for the UA case it was due to the surface area effect. Investigation with the standard bench mark redox system, Fe(CN)63, showed improper tail-off peak current behaviours due to diffusion restriction of the redox couple within the mesopores of the material. The effect of solution pH on the purine oxidation reactions obeyed the Nernstian type of proton-coupled electrontransfer reaction mechanism. Constructed calibration plots were linear up to 400, 320 and 240 mM of UA, X and HX respectively. Corresponding detection limit values were 110 nM, 388 nM and 351 nM. Simultaneous or selective detection of the three purine compounds in blood-plasma, healthy urine samples and sh samples was successfully demonstrated. A matrix effect was noticed with some real sample analyses. Calculated recovery
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